COMPOSITIONS AND METHODS FOR DETECTION OF BETA N METHYLAMINO L ALANINE IN CU ZN SUPEROXIDE DISMUTASE 1

Abstract

Provided herein are isotopically labeled reagents, including isotopically labeled small molecules and peptides, that can be used to detect and/or quantify .beta.-N-methylamino-L-alanine (BMAA) in the Cu/Zn Superoxide Dismutase 1 (SOD1) protein. Further provided are methods for detecting, preventing, or treating amyotrophic lateral sclerosis in a subject using isotopically labeled reagents to detect and/or quantify .beta.-N-methylamino-L-alanine (BMAA) in the Cu/Zn Superoxide Dismutase 1 (50D1) protein. Further provided are isotopically labeled reagents and methods for detecting BMAA in additional proteins from patient samples.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Ser. No. 62/368,437, filed Jul. 29, 2016, and U.S. Provisional Patent Application Ser. No. 62/368,562, filed Jul. 29, 2016, each of which are expressly incorporated herein by reference.

BACKGROUND

[0002] The non-protein amino acid .beta.-N-Methylamino-L-Alanine (BMAA) is biosynthetically produced in cyanobacteria. Human exposure to this unnatural amino acid has been linked to neurological disorders, including amyotrophic lateral sclerosis (ALS) and parkinsonism dementia complex (PDC) like symptoms. Dietary exposure of BMAA in primates has shown neurofibrillary tangles (NFT) and .beta.-amyloid plaques, hallmark signs of neuropathological disease. Additionally, cell culture studies have shown that exogenous exposure to BMAA can result in incorporation of this non-protein amino acid in place of L-serine. Supplemental treatment with L-serine has been shown to reduce the rate of BMAA incorporation and regression of neuropathological symptoms.

[0003] While these findings suggest that BMAA can be incorporated into proteins, efforts to further study the role of BMAA in biological systems has been hampered by the limited availability of suitable analytical probes and methods. Improved analytical tools and methods are needed to fully understand the qualitative and quantitative nature of incorporation of BMAA in proteins, particularly human proteins, as well as the mechanism and functional consequences of this process.

[0004] Because of the lack of appropriate reagents and methods, the identification of endogenous BMAA incorporation in patients suffering from neurological disorders, including amyotrophic lateral sclerosis (ALS), has not yet been shown. Thus, improved compositions and methods are needed for the detection of BMAA incorporation into proteins implicated in neurological disorders.

SUMMARY

[0005] Provided herein are isotopically labeled reagents, including isotopically labeled small molecules and peptides, that can be used to detect and/or quantify .beta.-N-methylamino-L-alanine (BMAA) in the Cu/Zn Superoxide Dismutase 1 (SOD1) protein. Using these novel reagents, the inventors have identified the incorporation of BMAA into the SOD1 protein in samples from patients suffering from amyotrophic lateral sclerosis (ALS). The reagents can be used as stable isotope labeled standards in analytical methods, including in conjunction with mass spectrometry, to detect and/or quantify BMAA in a sample, such as a protein sample from a subject. In addition, the inventors have developed isotopically labeled reagents that can detect the incorporation of BMAA into several additional proteins in patient samples.

[0006] In one aspect, provided herein is a polypeptide comprising a sequence that is at least 85% identical to SEQ ID NO: 1, wherein amino acid position 107 comprises a BMAA residue which is isotopically labeled and is defined by the formula below

##STR00001##

[0007] wherein the .sup.13C isotopic enrichment factor for .sup.dC is at least 25; and wherein the .sup.15N isotopic enrichment factor for N is at least 100.

[0008] In one aspect, provided herein is a polypeptide comprising a sequence that is at least 85% identical to SEQ ID NO: 2, wherein amino acid position 107 comprises a BMAA residue which is isotopically labeled and is defined by the formula below

##STR00002##

[0009] wherein the .sup.13C isotopic enrichment factor for .sup.dC is at least 25; and wherein the .sup.15N isotopic enrichment factor for N is at least 100.

[0010] In another aspect, provided herein is a method for detecting .beta.-N-methylamino-L-alanine (BMAA) in a SOD1 protein, comprising (a) purifying SOD1 protein from a biological specimen to provide a purified SOD1 protein sample, (b) spiking the purified SOD1 protein sample with a defined amount of an isotopically labeled compound defined by the formula below

##STR00003##

[0011] wherein R represents hydrogen or an amine protecting group, and at least two of .sup.aC, .sup.bC, .sup.cC, .sup.dC, .sup.eN, .sup.fN, .sup.gO, and .sup.hO are isotopically labeled with a stable isotope, to provide a BMAA-spiked sample; (c) analyzing the BMAA-spiked sample by mass spectrometry; and (d) measuring BMAA levels in the SOD1 protein sample by isotope dilution analysis.

[0012] In one aspect, provided herein is a method for detecting .beta.-N-methylamino-L-alanine (BMAA) in a SOD1 protein, comprising: (a) purifying SOD1 protein from a biological specimen to provide a purified SOD1 protein sample; (b) spiking the purified SOD1 protein sample with a defined amount of a polypeptide, wherein the polypeptide includes one or more isotopically labeled .beta.-N-methylamino-L-alanine (BMAA) residues, wherein each of the one or more isotopically labeled BMAA residues is isotopically labeled with one or more stable isotopes; to provide a BMAA-spiked sample; (c) analyzing the BMAA-spiked sample by mass spectrometry; and (d) measuring BMAA levels in the SOD1 protein sample by isotope dilution analysis.

[0013] In a further aspect, provided herein is a method of detecting or predicting amyotrophic lateral sclerosis in a subject, comprising (a) purifying SOD1 protein from a biological specimen from a subject to provide a purified SOD1 protein sample; (b) spiking the purified SOD1 protein sample with a defined amount of an isotopically labeled compound defined by the formula below

##STR00004##

[0014] wherein R represents hydrogen or an amine protecting group, and at least two of .sup.aC, .sup.bC, .sup.cC, .sup.dC, .sup.eN, .sup.fN, .sup.gO, and .sup.hO are isotopically labeled with a stable isotope, to provide a BMAA-spiked sample; (c) analyzing the BMAA-spiked sample by mass spectrometry; (d) measuring BMAA level in the SOD1 protein sample by isotope dilution analysis; and (e) identifying the subject for the presence or risk of amyotrophic lateral sclerosis if the BMAA level in the SOD1 protein is greater than a normal reference value.

[0015] In another aspect, provided herein is a method of detecting or predicting amyotrophic lateral sclerosis in a subject, comprising (a) purifying SOD1 protein from a biological specimen from a subject to provide a purified SOD1 protein sample; (b) spiking the purified SOD1 protein sample with a defined amount of a polypeptide, wherein the polypeptide includes one or more isotopically labeled .beta.-N-methylamino-L-alanine (BMAA) residues, wherein each of the one or more isotopically labeled BMAA residues is isotopically labeled with one or more stable isotopes, to provide a BMAA-spiked sample; (c) analyzing the BMAA-spiked sample by mass spectrometry; (d) measuring BMAA level in the SOD1 protein sample by isotope dilution analysis; and (e) identifying the subject for the presence or risk of amyotrophic lateral sclerosis if the BMAA level in the SOD1 protein is greater than a normal reference value.

[0016] In one aspect, provided herein is a method of preventing or treating amyotrophic lateral sclerosis in a subject, comprising (a) purifying SOD1 protein from a biological specimen from a subject to provide a purified SOD1 protein sample; (b) spiking the purified SOD1 protein sample with a defined amount of an isotopically labeled compound defined by the formula below

##STR00005##

[0017] wherein R represents hydrogen or an amine protecting group, and at least two of .sup.aC, .sup.bC, .sup.cC, .sup.dC, .sup.eN, .sup.fN, .sup.gO, and .sup.hO are isotopically labeled with a stable isotope, to provide a BMAA-spiked sample; (c) analyzing the BMAA-spiked sample by mass spectrometry; (d) measuring BMAA levels in the SOD1 protein sample by isotope dilution analysis; (e) identifying the subject for the presence or risk of amyotrophic lateral sclerosis if the BMAA levels in the SOD1 protein is greater than a normal reference value; and (f) administering to the subject L-serine in an amount sufficient to prevent or treat the amyotrophic lateral sclerosis.

[0018] In one final aspect, provided herein is a method of preventing or treating amyotrophic lateral sclerosis in a subject, comprising (a) purifying SOD1 protein from a biological specimen from a subject to provide a purified SOD1 protein sample; (b) spiking the purified SOD1 protein sample with a defined amount of a polypeptide, wherein the polypeptide includes one or more isotopically labeled .beta.-N-methylamino-L-alanine (BMAA) residues, wherein each of the one or more isotopically labeled BMAA residues is isotopically labeled with one or more stable isotopes, to provide a BMAA-spiked sample; (c) analyzing the BMAA-spiked sample by mass spectrometry; (d) measuring BMAA levels in the SOD1 protein sample by isotope dilution analysis; (e) identifying the subject for the presence or risk of amyotrophic lateral sclerosis if the BMAA levels in the SOD1 protein is greater than a normal reference value; and (f) administering to the subject L-serine in an amount sufficient to prevent or treat the amyotrophic lateral sclerosis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 is an illustration of an example procedure used to convert a phosphoserine-containing peptide to BMAA-containing peptide. The peptide sequence shown in FIG. 1 is unique to superoxide dismutase 1 (SOD1).

[0020] FIGS. 2A-2B illustrate the intact mass of stable isotope-labeled (SIL) phosphopeptide substrate and BMAA peptide product.

[0021] FIG. 3 illustrates the tandem mass spectrum of the phosphorylated serine-containing stable isotope-labeled peptide.

[0022] FIG. 4 illustrates the tandem mass spectrum of a BMAA-containing stable isotope-labeled peptide, confirming localization of modification.

[0023] FIG. 5 demonstrates that co-eluting fragments confirm identity of the BMAA-containing peptide. BMAA- and phosphoserine-containing peptides have distinct retention times. The BMAA-containing peptide appears to have two peaks which suggests L and D isomers of the BMAA peptide have been produced.

[0024] FIG. 6 illustrates the conserved peak area of fragments specific to SIL BMAA peptide that allow for confident identification of endogenous peptide.

[0025] FIG. 7 is a plot showing that the elution time of SIL BMAA peptide is necessary for correct identification of endogenous peptide. Without this, the incorrect peak which is also within 5 ppm mass accuracy of the BMAA peptide of interest might be selected.

[0026] FIG. 8 demonstrates the ability of mass spectrometry to accurately identify endogenous BMAA-containing peptide in SOD1 protein digest obtained from ALS erythrocytes.

[0027] FIG. 9 is a plot illustrating the co-elution of endogenous and stable isotope-labeled BMAA containing peptides. Co-elution aids in confident identification.

[0028] FIG. 10 is a schematic showing the method of filter aided sample preparation for bottom-up protein characterization.

[0029] FIG. 11 is a schematic showing the post-translational modifications identified with high confidence in SOD1. The amino acid numbering shown in the figure does not include the starting methionine.

[0030] FIG. 12 is a schematic showing the post-translational modifications identified with medium confidence in SOD1. The amino acid numbering shown in the figure does not include the starting methionine.

[0031] FIG. 13 illustrates the tandem mass spectrum of a BMAA-containing stable isotope-labeled peptide. Fragment ions corresponding to the endogenous peptide with BMAA were found at the expected location, confirming localization of modification.

[0032] FIG. 14 illustrates the tandem mass spectrum of BMAA containing stable isotope-label (.sup.13C.sub.4.sup.15N.sub.2 .beta.-N-Methylamino-L-Alanine, or Compound 3).

DETAILED DESCRIPTION

[0033] Provided herein are isotopically labeled reagents, including isotopically labeled small molecules and peptides, that can be used to detect and/or quantify .beta.-N-methylamino-L-alanine (BMAA) in the Cu/Zn Superoxide Dismutase 1 (SOD1) protein. Using these novel reagents, the inventors have identified the incorporation of endogenous BMAA into the SOD1 protein in samples from patients suffering from amyotrophic lateral sclerosis (ALS). The reagents can be used as stable isotope labeled standards in analytical methods, including in conjunction with mass spectrometry, to detect and/or quantify BMAA in a sample, such as a protein sample from a subject. In addition, the inventors have developed isotopically labeled reagents that can detect the incorporation of BMAA into several additional proteins in patient samples.

[0034] As used herein, "protein" and "polypeptide" are used synonymously to mean any peptide-linked chain of amino acids, regardless of length or post-translational modification, e.g., glycosylation or phosphorylation. Amino acids include Alanine (Ala, A), Asparagine (Asn, N), Cysteine (Cys, C), Glutamine (Gln, Q), Glycine (Gly, G), Isoleucine (Ile, I), Leucine (Leu, L), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), Valine (Val, V), Histidine (His, H), Arginine (Arg, R), Lysine (Lys, K), Aspartic acid (Asp, D), and Glutamic acid (Glu, E). .beta.-N-methylamino-L-alanine (BMAA) is represented using the letter B in a peptide sequence, unless otherwise indicated in the text.

[0035] As used herein, a "normal reference value" refers to a numerical value that is determined experimentally from an unaffected individual, or a population of unaffected individuals, for comparison to the value from an affected individual, or population of affected individuals (for example, individuals affected by ALS). In some embodiments, the "normal reference value" being measured is from a healthy control subject that is not affected by ALS, or from a population of healthy control subjects that are not affected by ALS.

Compounds and Compositions

[0036] Provided herein is a polypeptide comprising a sequence that is at least 85% identical to SEQ ID NO: 1, wherein amino acid position 107 comprises a BMAA residue which is isotopically labeled and is defined by the formula below

##STR00006##

[0037] wherein the .sup.13C isotopic enrichment factor for .sup.dC is at least 25; and wherein the .sup.15N isotopic enrichment factor for N is at least 100.

[0038] In one embodiment, the .sup.13C isotopic enrichment factor for .sup.dC is at least 80 and the .sup.15N isotopic enrichment factor for N is at least 200. In one embodiment, the .sup.13C isotopic enrichment factor for .sup.aC, .sup.bC, and .sup.cC is at least 25. In one embodiment, .sup.13C isotopic enrichment factor for .sup.aC, .sup.bC, and .sup.cC is at least 80. In one embodiment, .sup.15N isotopic enrichment factor for .sup.eN is at least 100. In one embodiment, .sup.15N isotopic enrichment factor for .sup.eN is at least 200.

[0039] In one embodiment, the sequence is at least 90% identical to SEQ ID NO: 1. In one embodiment, the sequence is at least 95% identical to SEQ ID NO: 1. In one embodiment, the sequence is at least 99% identical to SEQ ID NO: 1. In one embodiment, the sequence is identical to SEQ ID NO: 1.

[0040] In one aspect, provided herein is a polypeptide comprising a sequence that is at least 85% identical to SEQ ID NO: 2, wherein amino acid position 107 comprises a BMAA residue which is isotopically labeled and is defined by the formula below

##STR00007##

[0041] wherein the .sup.13C isotopic enrichment factor for .sup.dC is at least 25; and wherein the .sup.15N isotopic enrichment factor for N is at least 100.

[0042] In one embodiment, the .sup.13C isotopic enrichment factor for .sup.dC is at least 80 and the .sup.15N isotopic enrichment factor for N is at least 200. In one embodiment, the .sup.13C isotopic enrichment factor for .sup.aC, .sup.bC, and .sup.cC is at least 25. In one embodiment, the .sup.13C isotopic enrichment factor for .sup.aC, .sup.bC, and .sup.cC is at least 80. In one embodiment, the .sup.15N isotopic enrichment factor for .sup.eN is at least 100. In one embodiment, the .sup.15N isotopic enrichment factor for .sup.eN is at least 200.

[0043] In one embodiment, the sequence is at least 90% identical to SEQ ID NO: 2. In one embodiment, the sequence is at least 95% identical to SEQ ID NO: 2. In one embodiment, the sequence is at least 99% identical to SEQ ID NO: 2. In one embodiment, the sequence is identical to SEQ ID NO: 2.

[0044] In one embodiment, the sequence is at least 90% identical to SEQ ID NO: 3. In one embodiment, the sequence is at least 95% identical to SEQ ID NO: 3. In one embodiment, the sequence is at least 99% identical to SEQ ID NO: 3. In one embodiment, the sequence is identical to SEQ ID NO: 3.

TABLE-US-00001 SEQ ID NO: 1 = Human SOD1 protein sequence with BMAA at position 107 ATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEF GDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIE DSVISLBGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIG IAQ

Note that the BMAA sequence at position 107 (in bold) replaces the serine that is normally present in the wild type SOD1 sequence in SEQ ID NO: 1. The BMAA occurs at position 107 when the initial methionine is not included in the peptide sequence, but occurs at position 108 when the initial methionine is included in the sequence.

[0045] Additional sequences for polypeptides that can be used as reagents in methods for detection of BMAA incorporation into the SOD1 protein include:

TABLE-US-00002 SEQ ID NO: 2 = DGVADVSIEDSVISLBGDHCIIGR SEQ ID NO: 3 = HVGDLGNVTADKDGVADVSIEDSVISLBGDHCIIGR (1 missed cleavage) Where C is Carbamidomethylated Cysteine (alkylated during sample preparation) SEQ ID NO: 4 = Human SOD1 wild-type protein sequence. ATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEF GDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIE DSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIG IAQ SEQ ID NO: 5 = Human SOD1 protein sequence with BMAA at position 108 (with initial methionine) MATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHE FGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSI EDSVISLBGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVI GIAQ

Note that the BMAA sequence at position 108 (in bold) replaces the serine that is normally present in the wild type SOD1 sequence in SEQ ID NO: 5. The BMAA occurs at position 108 when the initial methionine is included in the peptide sequence, but occurs at position 107 when the initial methionine is not included in the sequence.

[0046] In one embodiment, the sequence is at least 90% identical to SEQ ID NO: 5. In one embodiment, the sequence is at least 95% identical to SEQ ID NO: 5. In one embodiment, the sequence is at least 99% identical to SEQ ID NO: 5. In one embodiment, the sequence is identical to SEQ ID NO: 5.

TABLE-US-00003 SEQ ID NO: 6 = Human SOD1 wild-type protein sequence (with initial methionine) MATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHE FGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSI EDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVI GIAQ

[0047] In one embodiment, the sequence is at least 90% identical to SEQ ID NO: 6. In one embodiment, the sequence is at least 95% identical to SEQ ID NO: 6. In one embodiment, the sequence is at least 99% identical to SEQ ID NO: 6. In one embodiment, the sequence is identical to SEQ ID NO: 6.

[0048] In certain embodiments, at least two (e.g., at least three, at least four, at least five, or all six) of .sup.aC, .sup.bC, .sup.cC, .sup.dC, .sup.eN, and .sup.fN are isotopically labeled with a stable isotope. In particular embodiments, all of .sup.aC, .sup.bC, .sup.cC, .sup.dC, .sup.eN, and .sup.fN are isotopically labeled with a stable isotope.

[0049] The term "isotopic enrichment factor," as used herein, refers to the ratio between the isotopic abundance (e.g., .sup.13C, .sup.15N, or .sup.18O) at a specified position in a compound and the naturally occurring abundance of that isotope. The naturally occurring abundance of .sup.13C is 1.1%. The naturally occurring abundance of .sup.15N is 0.37%. The naturally occurring abundance of .sup.18O is 0.204%.

[0050] In some embodiments, the .sup.13C isotopic enrichment factor for .sup.aC, .sup.bC, .sup.cC, and .sup.dC is at least 25 (27.5% .sup.13C incorporation at each position), at least 30 (33% .sup.13C incorporation at each position), at least 35 (38.5% .sup.13C incorporation at each position), at least 40 (44% .sup.13C incorporation at each position), at least 45 (49.5% .sup.13C incorporation at each position), at least 50 (55% .sup.13C incorporation at each position), at least 55 (60.5% .sup.13C incorporation at each position), at least 60 (66% .sup.13C incorporation at each position), at least 65 (71.5% .sup.13C incorporation at each position), at least 70 (77% .sup.13C incorporation at each position), at least 75 (82.5% .sup.13C incorporation at each position), at least 80 (88% .sup.13C incorporation at each position), at least 85 (93.5% .sup.13C incorporation at each position), or at least 90 (99% .sup.13C incorporation at each position).

[0051] In some embodiments, the .sup.15N isotopic enrichment factor for .sup.eN and .sup.fN is at least 100 (37% .sup.15N incorporation at each position), at least 110 (40.7% .sup.15N incorporation at each position), at least 120 (44.4% .sup.15N incorporation at each position), at least 130 (48.1% .sup.15N incorporation at each position), at least 140 (51.8% .sup.15N incorporation at each position), at least 150 (55.5% .sup.15N incorporation at each position), at least 160 (59.2% .sup.15N incorporation at each position), at least 170 (62.9% .sup.15N incorporation at each position), at least 180 (66.6% .sup.15N incorporation at each position), at least 190 (70.3% .sup.15N incorporation at each position), at least 200 (74% .sup.15N incorporation at each position), at least 210 (77.7% .sup.15N incorporation at each position), at least 220 (81.4% .sup.15N incorporation at each position), at least 230 (85.1% .sup.15N incorporation at each position), at least 240 (88.8% .sup.15N incorporation at each position), at least 250 (92.5% .sup.15N incorporation at each position), at least 260 (96.2% .sup.15N incorporation at each position), or at least 265 (98.05% .sup.15N incorporation at each position).

[0052] In some embodiments, the polypeptides can be comprised in a composition. The composition can be, for example, a solution of the isotopically labeled compound in a solvent. Non-limiting examples of solvents include aliphatic solvents (e.g., pentane, hexanes, cyclohexane); aromatic and/or alkylated aromatic solvents such as benzene, toluene, xylene; hydrocarbon solvents; dichloromethane, chloroform, alcohols (e.g., methanol, ethanol, isopropanol); esters (e.g., ethyl acetate); ketones (e.g., acetone); diethyl ether; dioxane; glycol ethers and glycol ether esters; tetrahydrofuran; dimethylformamide; acetonitrile; dimethyl sulfoxide; water, saline, aqueous buffers (e.g., PBS buffer), and combinations thereof. In certain examples, the composition can comprise an aqueous solution of the compound.

[0053] In some embodiments, the isotopically labeled compound can comprise at least 0.5% by weight (e.g., at least 1% by weight, at least 1.5% by weight, at least 2% by weight, at least 2.5% by weight, at least 3% by weight, at least 3.5% by weight, at least 4% by weight, at least 4.5% by weight, or at least 1% by weight of the composition.

[0054] The isotopically labeled compounds described above can be prepared using methods known in the art. Representative methodologies for the preparation of certain active agents are described below. The appropriate route for synthesis of a given compound agent can be selected in view of the structure of the compound as a whole as it relates to compatibility of functional groups, protecting group strategies, and the presence of labile bonds. In addition to the synthetic methodologies discussed below, alternative reactions and strategies useful for the preparation of the compounds disclosed herein are known in the art. See, for example, March, "Advanced Organic Chemistry," 5.sup.th Edition, 2001, Wiley-Interscience Publication, New York).

[0055] Isotopically labeled amino acids, such as .sup.13C/.sup.15N-labeled asparagine, are commercially available, and can serve as convenient starting materials for the isotopically labeled compounds described herein. Scheme 1 below illustrates an example method for the preparation of BMAA from asparagine. Compounds having a desired isotopic labeling (e.g., incorporating stable isotopes at particular positions within the compound) can be prepared by selecting reagents that include stable isotope labels at the appropriate positions with their framework (e.g., .sup.13C/.sup.15N-labeled asparagine).

[0056] In some embodiments, the BMAA amino acid residue is isotopically labeled. In some embodiments, the BMAA amino acid residue is unlabeled and the peptide is isotopically labeled at an amino acid other than at the BMAA residue.

[0057] In some embodiments, provided herein is a polypeptide comprising a sequence that is at least 85% identical to SEQ ID NO: 1, wherein amino acid position 107 comprises a BMAA residue, wherein the polypeptide is isotopically labeled. In some embodiments, provided herein is a polypeptide comprising a sequence that is at least 85% identical to SEQ ID NO: 1, wherein amino acid position 107 comprises a BMAA residue, wherein the polypeptide is isotopically labeled and the isotopic label is enriched in comparison to the corresponding naturally occurring polypeptide.

##STR00008##

[0058] Also provided are compositions that include one or more isotopically labeled SOD1 polypeptides of SOD1 peptide fragments. For example, compositions comprising a SOD1 polypeptide that includes one or more isotopically labeled .beta.-N-methylamino-L-alanine (BMAA) residues are provided herein. The isotopically labeled SOD1 polypeptide can comprise at least 0.5% by weight of the composition. Each of the one or more isotopically labeled BMAA residues can be isotopically labeled with one or more (e.g., two or more) stable isotopes. For example, each of the one or more isotopically labeled BMAA residues can be defined by the formula below

##STR00009##

where the .sup.13C isotopic enrichment factor for .sup.dC is at least 25; and the .sup.15N isotopic enrichment factor for .sup.fN is at least 100.

[0059] In some embodiments, the .sup.13C isotopic enrichment factor for .sup.dC is at least 25 (27.5% .sup.13C incorporation at each position), at least 30 (33% .sup.13C incorporation at each position), at least 35 (38.5% .sup.13C incorporation at each position), at least 40 (44% .sup.13C incorporation at each position), at least 45 (49.5% .sup.13C incorporation at each position), at least 50 (55% .sup.13C incorporation at each position), at least 55 (60.5% .sup.13C incorporation at each position), at least 60 (66% .sup.13C incorporation at each position), at least 65 (71.5% .sup.13C incorporation at each position), at least 70 (77% .sup.13C incorporation at each position), at least 75 (82.5% .sup.13C incorporation at each position), at least 80 (88% .sup.13C incorporation at each position), at least 85 (93.5% .sup.13C incorporation at each position), or at least 90 (99% .sup.13C incorporation at each position).

[0060] In some embodiments, the .sup.15N isotopic enrichment factor for .sup.fN is at least 100 (37% .sup.15N incorporation at each position), at least 110 (40.7% .sup.15N incorporation at each position), at least 120 (44.4% .sup.15N incorporation at each position), at least 130 (48.1% .sup.15N incorporation at each position), at least 140 (51.8% .sup.15N incorporation at each position), at least 150 (55.5% .sup.15N incorporation at each position), at least 160 (59.2% .sup.15N incorporation at each position), at least 170 (62.9% .sup.15N incorporation at each position), at least 180 (66.6% .sup.15N incorporation at each position), at least 190 (70.3% .sup.15N incorporation at each position), at least 200 (74% .sup.15N incorporation at each position), at least 210 (77.7% .sup.15N incorporation at each position), at least 220 (81.4% .sup.15N incorporation at each position), at least 230 (85.1% .sup.15N incorporation at each position), at least 240 (88.8% .sup.15N incorporation at each position), at least 250 (92.5% .sup.15N incorporation at each position), at least 260 (96.2% .sup.15N incorporation at each position), or at least 265 (98.05% .sup.15N incorporation at each position).

[0061] Optionally, .sup.aC, .sup.bC, and/or .sup.cC can also be labeled with a stable isotope. In some cases, the .sup.13C isotopic enrichment factor for .sup.aC, .sup.bC, and .sup.cC can be at least 25 (27.5% .sup.13C incorporation at each position), at least 30 (33% .sup.13C incorporation at each position), at least 35 (38.5% .sup.13C incorporation at each position), at least 40 (44% .sup.13C incorporation at each position), at least 45 (49.5% .sup.13C incorporation at each position), at least 50 (55% .sup.13C incorporation at each position), at least 55 (60.5% .sup.13C incorporation at each position), at least 60 (66% .sup.13C incorporation at each position), at least 65 (71.5% .sup.13C incorporation at each position), at least 70 (77% .sup.13C incorporation at each position), at least 75 (82.5% .sup.13C incorporation at each position), at least 80 (88% .sup.13C incorporation at each position), at least 85 (93.5% .sup.13C incorporation at each position), or at least 90 (99% .sup.13C incorporation at each position).

[0062] Optionally, .sup.eN can also be labeled with a stable isotope. In some cases, the .sup.15N isotopic enrichment factor for .sup.eN is at least 100 (37% .sup.15N incorporation at each position), at least 110 (40.7% .sup.15N incorporation at each position), at least 120 (44.4% .sup.15N incorporation at each position), at least 130 (48.1% .sup.15N incorporation at each position), at least 140 (51.8% .sup.15N incorporation at each position), at least 150 (55.5% .sup.15N incorporation at each position), at least 160 (59.2% .sup.15N incorporation at each position), at least 170 (62.9% .sup.15N incorporation at each position), at least 180 (66.6% .sup.15N incorporation at each position), at least 190 (70.3% .sup.15N incorporation at each position), at least 200 (74% .sup.15N incorporation at each position), at least 210 (77.7% .sup.15N incorporation at each position), at least 220 (81.4% .sup.15N incorporation at each position), at least 230 (85.1% .sup.15N incorporation at each position), at least 240 (88.8% .sup.15N incorporation at each position), at least 250 (92.5% .sup.15N incorporation at each position), at least 260 (96.2% .sup.15N incorporation at each position), or at least 265 (98.05% .sup.15N incorporation at each position).

[0063] In some embodiments, the polypeptide can include a single isotopically labeled BMAA residue. In other embodiments, the polypeptide can include two or more isotopically labeled BMAA residues (e.g., three or more isotopically labeled BMAA residues, four or more isotopically labeled BMAA residues, five or more isotopically labeled BMAA residues, or ten or more isotopically labeled BMAA residues). In some embodiments, the BMAA residue is substituted for Ser107 (numbering does not include the starting methionine.

[0064] In some embodiments, the polypeptide can be defined by the formula below

##STR00010##

where m is an integer from 0 to 8384 and n is an integer from 0 to 8384, with the proviso that at least one of m and n is not 0; the .sup.13C isotopic enrichment factor for .sup.dC is at least 25; the .sup.15N isotopic enrichment factor for N is at least 100; and independently for each occurrence in the polypeptide, R.sub.1 is H and R.sub.2 is selected from one of the following

##STR00011##

or R.sub.1 and R.sub.2, together with the atoms to which they are attached, form a five-membered heterocycle defined by the structure below

##STR00012##

[0065] In some embodiments, the polypeptide can be defined by the formula below

##STR00013##

where m is an integer from 0 to 108 and n is an integer from 0 to 46, with the proviso that at least one of m and n is not 0; the .sup.13C isotopic enrichment factor for .sup.dC is at least 25; the .sup.15N isotopic enrichment factor for N is at least 100; and independently for each occurrence in the polypeptide, R.sub.1 is H and R.sub.2 is selected from one of the following

##STR00014##

or R.sub.1 and R.sub.2, together with the atoms to which they are attached, form a five-membered heterocycle defined by the structure below

##STR00015##

[0066] In some embodiments, the .sup.13C isotopic enrichment factor for .sup.dC is at least 25 (27.5% .sup.13C incorporation at each position), at least 30 (33% .sup.13C incorporation at each position), at least 35 (38.5% .sup.13C incorporation at each position), at least 40 (44% .sup.13C incorporation at each position), at least 45 (49.5% .sup.13C incorporation at each position), at least 50 (55% .sup.13C incorporation at each position), at least 55 (60.5% .sup.13C incorporation at each position), at least 60 (66% .sup.13C incorporation at each position), at least 65 (71.5% .sup.13C incorporation at each position), at least 70 (77% .sup.13C incorporation at each position), at least 75 (82.5% .sup.13C incorporation at each position), at least 80 (88% .sup.13C incorporation at each position), at least 85 (93.5% .sup.13C incorporation at each position), or at least 90 (99% .sup.13C incorporation at each position).

[0067] In some embodiments, the .sup.15N isotopic enrichment factor for .sup.fN is at least 100 (37% .sup.15N incorporation at each position), at least 110 (40.7% .sup.15N incorporation at each position), at least 120 (44.4% .sup.15N incorporation at each position), at least 130 (48.1% .sup.15N incorporation at each position), at least 140 (51.8% .sup.15N incorporation at each position), at least 150 (55.5% .sup.15N incorporation at each position), at least 160 (59.2% .sup.15N incorporation at each position), at least 170 (62.9% .sup.15N incorporation at each position), at least 180 (66.6% .sup.15N incorporation at each position), at least 190 (70.3% .sup.15N incorporation at each position), at least 200 (74% .sup.15N incorporation at each position), at least 210 (77.7% .sup.15N incorporation at each position), at least 220 (81.4% .sup.15N incorporation at each position), at least 230 (85.1% .sup.15N incorporation at each position), at least 240 (88.8% .sup.15N incorporation at each position), at least 250 (92.5% .sup.15N incorporation at each position), at least 260 (96.2% .sup.15N incorporation at each position), or at least 265 (98.05% .sup.15N incorporation at each position).

[0068] Optionally, .sup.aC, .sup.bC, and/or .sup.cC can also be labeled with a stable isotope. In some cases, the .sup.13C isotopic enrichment factor for .sup.aC, .sup.bC, and .sup.cC can be at least 25 (27.5% .sup.13C incorporation at each position), at least 30 (33% .sup.13C incorporation at each position), at least 35 (38.5% .sup.13C incorporation at each position), at least 40 (44% .sup.13C incorporation at each position), at least 45 (49.5% .sup.13C incorporation at each position), at least 50 (55% .sup.13C incorporation at each position), at least 55 (60.5% .sup.13C incorporation at each position), at least 60 (66% .sup.13C incorporation at each position), at least 65 (71.5% .sup.13C incorporation at each position), at least 70 (77% .sup.13C incorporation at each position), at least 75 (82.5% .sup.13C incorporation at each position), at least 80 (88% .sup.13C incorporation at each position), at least 85 (93.5% .sup.13C incorporation at each position), or at least 90 (99% .sup.13C incorporation at each position).

[0069] Optionally, .sup.eN can also be labeled with a stable isotope. In some cases, the .sup.15N isotopic enrichment factor for .sup.eN is at least 100 (37% .sup.15N incorporation at each position), at least 110 (40.7% .sup.15N incorporation at each position), at least 120 (44.4% .sup.15N incorporation at each position), at least 130 (48.1% .sup.15N incorporation at each position), at least 140 (51.8% .sup.15N incorporation at each position), at least 150 (55.5% .sup.15N incorporation at each position), at least 160 (59.2% .sup.15N incorporation at each position), at least 170 (62.9% .sup.15N incorporation at each position), at least 180 (66.6% .sup.15N incorporation at each position), at least 190 (70.3% .sup.15N incorporation at each position), at least 200 (74% .sup.15N incorporation at each position), at least 210 (77.7% .sup.15N incorporation at each position), at least 220 (81.4% .sup.15N incorporation at each position), at least 230 (85.1% .sup.15N incorporation at each position), at least 240 (88.8% .sup.15N incorporation at each position), at least 250 (92.5% .sup.15N incorporation at each position), at least 260 (96.2% .sup.15N incorporation at each position), or at least 265 (98.05% .sup.15N incorporation at each position).

[0070] In some embodiments, m can be at least 1 (e.g., at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100). In some embodiments, m can be 108 or less (e.g., 100 or less, 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 or less, 20 or less, 10 or less, or 5 or less). m can be an integer ranging from any of the minimum values described above to any of the maximum values described above. For example, in some embodiments, m can be an integer from 1 to 108 (e.g., from 1 to 100, from 1 to 50, from 1 to 30, or from 1 to 10).

[0071] In some embodiments, n can be at least 1 (e.g., at least 5, at least 10, at least 20, at least 30, at least 40). In some embodiments, n can be 46 or less (e.g., 40 or less, 30 or less, 20 or less, 10 or less, or 5 or less). n can be an integer ranging from any of the minimum values described above to any of the maximum values described above. For example, in some embodiments, n can be an integer from 1 to 46 (e.g., from 1 to 40, from 1 to 30, from 1 to 20, or from 1 to 10).

[0072] In some embodiments, the sum of m and n can be at least 1 (e.g., at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150). In some embodiments, the sum of m and n can be 154 or less (e.g., 150 or less, 140 or less, 130 or less, 120 or less, 110 or less, 100 or less, 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 or less, 20 or less, 10 or less, or 5 or less). The sum of m and n can range from any of the minimum values described above to any of the maximum values described above. For example, in some embodiments, the sum of m and n can be from 1 to 154 (e.g., from 1 to 150, from 1 to 100, from 1 to 50, from 1 to 30, from 1 to 10, from 5 to 150, from 5 to 100, from 5 to 50, from 5 to 30, or from 5 to 10).

[0073] The composition can be, for example, a solution of the isotopically labeled peptide in a solvent. Non-limiting examples of solvents include alcohols (e.g., methanol, ethanol, isopropanol); esters (e.g., ethyl acetate); ketones (e.g., acetone); diethyl ether; dioxane; glycol ethers and glycol ether esters; tetrahydrofuran; dimethylformamide; acetonitrile; dimethyl sulfoxide; water, saline, aqueous buffers (e.g., PBS buffer), and combinations thereof. In certain examples, the composition can comprise an aqueous solution of the peptide.

[0074] In some embodiments, the isotopically labeled peptide can comprise at least 0.5% by weight (e.g., at least 1% by weight, at least 1.5% by weight, at least 2% by weight, at least 2.5% by weight, at least 3% by weight, at least 3.5% by weight, at least 4% by weight, at least 4.5% by weight, or at least 1% by weight of the composition.

[0075] Also provided are compositions comprising an isotopically labeled polypeptide that includes one or more .beta.-N-methylamino-L-alanine (BMAA) residues (e.g., one or more BMAA residues that are not isotopically labeled) and one or more additional amino acid residues that are labeled with one or more stable isotopes (.sup.13C, .sup.15N, and/or .sup.18O). In some embodiments, each residue labeled with one or more stable isotopes in the polypeptide includes at least two (e.g., at least four, at least five, at least six, or more) stable isotopes. For example, at least two (e.g., at least four, at least five, at least six, or more) of the carbon, nitrogen, and/or oxygen atoms in the residue can be isotopically labeled with a stable isotope. In some cases, at least two (e.g., at least four, at least five, at least six, or more) of the carbon and/or nitrogen atoms in the residue can be isotopically labeled with a stable isotope. The isotopically labeled polypeptide can comprise at least 0.5% by weight of the composition.

[0076] In some embodiments, the polypeptide can include a single isotopically labeled residue. In other embodiments, the polypeptide can include two or more isotopically labeled residues (e.g., three or more isotopically labeled residues, four or more isotopically labeled residues, five or more isotopically labeled residues, or ten or more isotopically labeled residues). In some embodiments, the polypeptide can include a single BMAA residue. In other embodiments, the polypeptide can include two or more BMAA residues (e.g., three or more BMAA residues, four or more BMAA residues, five or more BMAA residues, or ten or more BMAA residues).

[0077] In certain embodiments, the isotopically labeled polypeptide can be a peptide that includes one or more BMAA residues and a terminal amino acid residue (e.g., a terminal arginine residue) labeled with one or more stable isotopes (.sup.13C, .sup.15N, and/or .sup.18O).

[0078] The composition can be, for example, a solution of the isotopically labeled peptide in a solvent. Non-limiting examples of solvents include alcohols (e.g., methanol, ethanol, isopropanol); esters (e.g., ethyl acetate); ketones (e.g., acetone); diethyl ether; dioxane; glycol ethers and glycol ether esters; tetrahydrofuran; dimethylformamide; acetonitrile; dimethyl sulfoxide; water, saline, aqueous buffers (e.g., PBS buffer), and combinations thereof. In certain examples, the composition can comprise an aqueous solution of the peptide.

[0079] In some embodiments, the isotopically labeled peptide can comprise at least 0.5% by weight (e.g., at least 1% by weight, at least 1.5% by weight, at least 2% by weight, at least 2.5% by weight, at least 3% by weight, at least 3.5% by weight, at least 4% by weight, at least 4.5% by weight, or at least 1% by weight of the composition.

[0080] The peptides described above can be above can be prepared using a variety of methods known in the art. For example, peptides can be prepared using the isotopically labeled compounds described herein via solid phase peptide synthesis. The proteins and peptides described above can also be prepared by chemical derivatization of one or more residues within the protein and peptide. For example, a protein or peptide having a isotopically labeled .beta.-N-methylamino-L-alanine (BMAA) residue can be prepared from a protein or peptide that includes a phosphoserine residue. The protein or peptide can be reacted to convert the phosphoserine residue to an .alpha.,.beta.-unsaturated amino acid residue. Once activated, the .alpha.,.beta.-unsaturated amino acid residue can be reacted with methylamine (e.g., methylamine that is isotopically enriched with one or more stable isotopes), which undergoes a Michael-type addition to afford an isotopically labeled BMAA residue. This can involve, for example, reaction of the protein or peptide with methylamine-HCl (e.g., 1.0 M .sup.13C/.sup.15N-labeled methylamine) and Ba(OH).sub.2 (e.g., 0.1 M Ba(OH).sub.2) in water/DMSO/EtOH (2:2:1) at basic pH (pH 12.5) and elevated temperature (e.g., 37.degree. C.). Once complete, the reaction can be quenched with acid (e.g., acetic acid).

[0081] Full length natural or stable isotope labeled proteins comprising stable isotope labeled BMAA residues (e.g., incorporated at one or more specific sites within the protein) can be prepared by first preparing a phosphoserine-containing protein using an amber stop codon and a tRNA synthetase engineered to incorporate phosphoserine within the desired protein. See, for example, Rogerson, D. T. et al. "Efficient genetic encoding of phosphoserine and its nonhydrolyzable analog." Nat. Chem. Biol., 2015, 7: 496-503. The protein can then be isolated, and the phosphoserine can be chemically converted into BMAA using the strategy described above.

[0082] Further provided herein are isotopically labeled reagents, including isotopically labeled small molecules and peptides, that can be used to detect and/or quantify .beta.-N-methylamino-L-alanine (BMAA) in a protein selected from the validated targets in Table 6. Using these novel reagents, the inventors have identified the incorporation of BMAA into numerous proteins in samples from patients suffering from amyotrophic lateral sclerosis (ALS) and from control samples. The reagents can be used as stable isotope labeled standards in analytical methods, including in conjunction with mass spectrometry, to detect and/or quantify BMAA in a sample, such as a protein sample from a subject.

[0083] In one aspect, provided herein is a polypeptide comprising a sequence that is at least 85% identical a sequence selected from SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, or SEQ ID NO:49, wherein at least one amino acid comprises a BMAA residue which is isotopically labeled and is defined by the formula below

##STR00016##

[0084] wherein the .sup.13C isotopic enrichment factor for .sup.dC is at least 25; and wherein the .sup.15N isotopic enrichment factor for .sup.fN is at least 100.

[0085] In one aspect, provided herein is a polypeptide comprising a sequence selected from SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, or SEQ ID NO:49, wherein at least one amino acid comprises a BMAA residue which is isotopically labeled and is defined by the formula below

##STR00017##

[0086] wherein the .sup.13C isotopic enrichment factor for .sup.dC is at least 25; and wherein the .sup.15N isotopic enrichment factor for .sup.fN is at least 100.

[0087] In one aspect, provided herein is a polypeptide comprising a sequence that is at least 85% identical a sequence selected from SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75, wherein at least one amino acid comprises a BMAA residue which is isotopically labeled and is defined by the formula below

##STR00018##

[0088] wherein the .sup.13C isotopic enrichment factor for .sup.dC is at least 25; and wherein the .sup.15N isotopic enrichment factor for N is at least 100.

[0089] In one aspect, provided herein is a polypeptide comprising a sequence selected from SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75, wherein at least one amino acid comprises a BMAA residue which is isotopically labeled and is defined by the formula below

##STR00019##

[0090] wherein the .sup.13C isotopic enrichment factor for .sup.dC is at least 25; and wherein the .sup.15N isotopic enrichment factor for N is at least 100.

Methods

[0091] The compounds, peptides, proteins, and compositions described herein can be used detect and/or quantify BMAA in a SOD1 protein from a biological specimen (e.g., a sample from a patient suffering from neurodegenerative disease such as ALS), to accurately monitor BMAA exposure, to direct therapies, and in clinical diagnosis and prognosis.

[0092] For example, the isotopically labeled reagents and compositions described herein can be used in a variety of analytical methods to detect and/or quantify BMAA, such as to detect and/or quantify BMAA in a biological specimen or sample such as a protein sample. The isotopically labeled compounds described herein can be used to quantify free BMAA (e.g., quantify free BMAA in an environmental sample or biological sample), quantify total levels of BMAA in a protein sample (for example, a SOD1 protein sample), and/or to quantify protein-specific BMAA incorporation (e.g., by upstream purification of the protein of interest prior to analysis). The isotopically labeled compounds described herein can also be utilized in a stable isotope labeling by amino acids in cell culture (SILAC) alone or in combination with other stable isotope labeled amino acids. The isotopically labeled compounds described herein provide many analytical advantages over potential alternatives, such as deuterated BMAA, which does not co-elute and can undergo hydrogen-deuterium exchange in-solution or in the gas phase, significantly impacting identification and quantitation.

[0093] In one aspect, provided herein is a method for detecting .beta.-N-methylamino-L-alanine (BMAA) in a SOD1 protein, comprising (a) purifying SOD1 protein from a biological specimen to provide a purified SOD1 protein sample, (b) spiking the purified SOD1 protein sample with a defined amount of an isotopically labeled compound defined by the formula below

##STR00020##

[0094] wherein R represents hydrogen or an amine protecting group, and at least two of .sup.aC, .sup.bC, .sup.cC, .sup.dC, .sup.eN, .sup.fN, .sup.gO, and .sup.hO are isotopically labeled with a stable isotope, to provide a BMAA-spiked sample; (c) analyzing the BMAA-spiked sample by mass spectrometry; and (d) measuring BMAA levels in the SOD1 protein sample by isotope dilution analysis.

[0095] In one embodiment, at least three of .sup.aC, .sup.bC, .sup.cC, .sup.dC, .sup.eN, .sup.fN, .sup.gO, and .sup.hO are isotopically labeled with a stable isotope. In one embodiment, at least four of .sup.aC, .sup.bC, .sup.cC, .sup.dC, .sup.eN, .sup.fN, .sup.gO, and .sup.hO are isotopically labeled with a stable isotope. In one embodiment, at least three of .sup.aC, .sup.bC, .sup.cC, .sup.dC, .sup.eN, and .sup.fN are isotopically labeled with a stable isotope. In one embodiment, at least four of .sup.aC, .sup.bC, .sup.cC, .sup.dC, .sup.eN, and .sup.fN are isotopically labeled with a stable isotope. In one embodiment, .sup.aC, .sup.bC, .sup.cC, .sup.dC, .sup.eN, and .sup.fN are isotopically labeled with a stable isotope.

[0096] In one embodiment, the isotopically labeled compound is defined by the formula below

##STR00021##

[0097] wherein R represents hydrogen or an amine protecting group; wherein the .sup.13C isotopic enrichment factor for .sup.aC, .sup.bC, .sup.cC, and .sup.dC is at least 25; and wherein the .sup.15N isotopic enrichment factor for .sup.eN and .sup.fN is at least 100.

[0098] In one embodiment, the .sup.13C isotopic enrichment factor for .sup.aC, .sup.bC, .sup.cC, and .sup.dC is at least 80 and the .sup.15N isotopic enrichment factor for .sup.eN and .sup.fN is at least 200. In one embodiment, R is hydrogen. In one embodiment, R represents a 9-fluorenylmethyloxycarbonyl group.

[0099] On one embodiment, the purified SOD1 protein sample is hydrolyzed into amino acids for free BMAA analysis of SOD1.

[0100] In one aspect, provided herein is a method for detecting .beta.-N-methylamino-L-alanine (BMAA) in a SOD1 protein, comprising: (a) purifying SOD1 protein from a biological specimen to provide a purified SOD1 protein sample; (b) spiking the purified SOD1 protein sample with a defined amount of a polypeptide, wherein the polypeptide includes one or more isotopically labeled .beta.-N-methylamino-L-alanine (BMAA) residues, wherein each of the one or more isotopically labeled BMAA residues is isotopically labeled with one or more stable isotopes; to provide a BMAA-spiked sample; (c) analyzing the BMAA-spiked sample by mass spectrometry; and (d) measuring BMAA levels in the SOD1 protein sample by isotope dilution analysis.

[0101] In one embodiment, each of the one or more isotopically labeled BMAA residues is isotopically labeled with two or more stable isotopes. In one embodiment, each of the one or more isotopically labeled BMAA residues is defined by the formula below

##STR00022##

[0102] wherein the .sup.13C isotopic enrichment factor for .sup.dC is at least 25; and wherein the .sup.15N isotopic enrichment factor for .sup.fN is at least 100.

[0103] In one embodiment, the .sup.13C isotopic enrichment factor for .sup.dC is at least 80 and the .sup.15N isotopic enrichment factor for .sup.fN is at least 200. In one embodiment, the .sup.13C isotopic enrichment factor for .sup.aC, .sup.bC, and .sup.cC is at least 25. In one embodiment, the .sup.13C isotopic enrichment factor for .sup.aC, .sup.bC, and .sup.cC is at least 80. In one embodiment, the .sup.15N isotopic enrichment factor for .sup.eN is at least 100. In one embodiment, the .sup.15N isotopic enrichment factor for .sup.eN is at least 200.

[0104] In one embodiment, the polypeptide comprises a sequence is at least 85% identical to SEQ ID NO: 1. In one embodiment, the polypeptide comprises a sequence is at least 90% identical to SEQ ID NO: 1. In one embodiment, the polypeptide comprises a sequence is at least 95% identical to SEQ ID NO: 1. In one embodiment, the polypeptide comprises a sequence is at least 99% identical to SEQ ID NO: 1. In one embodiment, the polypeptide comprises a sequence is identical to SEQ ID NO: 1. In one embodiment, the polypeptide comprises a sequence is at least 85% identical to SEQ ID NO: 2. In one embodiment, the polypeptide comprises a sequence is at least 90% identical to SEQ ID NO: 2. In one embodiment, the polypeptide comprises a sequence is at least 95% identical to SEQ ID NO: 2. In one embodiment, the polypeptide comprises a sequence is at least 99% identical to SEQ ID NO: 2. In one embodiment, the polypeptide comprises a sequence is identical to SEQ ID NO: 2. In one embodiment, the purified SOD1 protein sample is digested with trypsin.

[0105] In one aspect, provided herein is a method of detecting or predicting amyotrophic lateral sclerosis in a subject, comprising (a) purifying SOD1 protein from a biological specimen from a subject to provide a purified SOD1 protein sample; (b) spiking the purified SOD1 protein sample with a defined amount of an isotopically labeled compound defined by the formula below

##STR00023##

[0106] wherein R represents hydrogen or an amine protecting group, and at least two of .sup.aC, .sup.bC, .sup.cC, .sup.dC, .sup.eN, .sup.fN, .sup.gO, and .sup.hO are isotopically labeled with a stable isotope, to provide a BMAA-spiked sample; (c) analyzing the BMAA-spiked sample by mass spectrometry; (d) measuring BMAA level in the SOD1 protein sample by isotope dilution analysis; and (e) identifying the subject for the presence or risk of amyotrophic lateral sclerosis if the BMAA level in the SOD1 protein is greater than a normal reference value.

[0107] In one embodiment, at least three of .sup.aC, .sup.bC, .sup.cC, .sup.dC, .sup.eN, .sup.fN, .sup.gO, and .sup.hO are isotopically labeled with a stable isotope. In one embodiment, at least four of .sup.aC, .sup.bC, .sup.cC, .sup.dC, .sup.eN, .sup.fN, .sup.gO, and .sup.hO are isotopically labeled with a stable isotope. In one embodiment, at least three of .sup.aC, .sup.bC, .sup.cC, .sup.dC, .sup.eN, and .sup.fN are isotopically labeled with a stable isotope. In one embodiment, at least four of .sup.aC, .sup.bC, .sup.cC, .sup.dC, .sup.eN, and .sup.fN are isotopically labeled with a stable isotope. In one embodiment, .sup.aC, .sup.bC, .sup.cC, .sup.dC, .sup.eN, and .sup.fN are isotopically labeled with a stable isotope. In one embodiment, the isotopically labeled compound is defined by the formula below

##STR00024##

[0108] wherein R represents hydrogen or an amine protecting group; wherein the .sup.13C isotopic enrichment factor for .sup.aC, .sup.bC, .sup.cC, and .sup.dC is at least 25; and wherein the .sup.15N isotopic enrichment factor for .sup.eN and .sup.fN is at least 100. In one embodiment, the .sup.13C isotopic enrichment factor for .sup.aC, .sup.bC, .sup.cC, and .sup.dC is at least 80 and the .sup.15N isotopic enrichment factor for .sup.eN and .sup.fN is at least 200. In one embodiment, R is hydrogen. In one embodiment, R represents a 9-fluorenylmethyloxycarbonyl group.

[0109] In one embodiment, the SOD1 protein further comprises a post-translational modification selected from the group consisting of a phospho group, acetyl group, glutathione, crotonaldehyde, glyoxal derived carboxymethyllysine, carboxyethyl, propionyl, crotonaldehyde derived dimethyl-FDP-lysine, 4-hydroxynonenal sulfonation and nitrosylation. In one embodiment, the SOD1 protein further comprises a post-translational modification selected from the group consisting of a phospho group, acetyl group, glutathione, and crotonaldehyde.

[0110] In one embodiment, the subject being identified for the presence or risk of amyotrophic lateral sclerosis comprises a BMAA level in the SOD1 protein that is at least 10% greater (e.g. at least 10% greater, at least 20% greater, at least 50% greater, at least 100% greater, or more) than the normal reference value.

[0111] In one aspect, provided herein is a method of detecting or predicting amyotrophic lateral sclerosis in a subject, comprising (a) purifying SOD1 protein from a biological specimen from a subject to provide a purified SOD1 protein sample; (b) spiking the purified SOD1 protein sample with a defined amount of a polypeptide, wherein the polypeptide includes one or more isotopically labeled .beta.-N-methylamino-L-alanine (BMAA) residues, wherein each of the one or more isotopically labeled BMAA residues is isotopically labeled with one or more stable isotopes, to provide a BMAA-spiked sample; (c) analyzing the BMAA-spiked sample by mass spectrometry; (d) measuring BMAA level in the SOD1 protein sample by isotope dilution analysis; and (e) identifying the subject for the presence or risk of amyotrophic lateral sclerosis if the BMAA level in the SOD1 protein is greater than a normal reference value.

[0112] In one embodiment, each of the one or more isotopically labeled BMAA residues is isotopically labeled with two or more stable isotopes. In one embodiment, each of the one or more isotopically labeled BMAA residues is defined by the formula below

##STR00025##

[0113] wherein the .sup.13C isotopic enrichment factor for .sup.dC is at least 25; and wherein the .sup.15N isotopic enrichment factor for .sup.fN is at least 100. In one embodiment, the .sup.13C isotopic enrichment factor for .sup.dC is at least 80 and the .sup.15N isotopic enrichment factor for .sup.fN is at least 200. In one embodiment, the .sup.13C isotopic enrichment factor for .sup.aC, .sup.bC, and .sup.cC is at least 25. In one embodiment, the .sup.13C isotopic enrichment factor for .sup.aC, .sup.bC, and .sup.cC is at least 80. In one embodiment, the .sup.15N isotopic enrichment factor for .sup.eN is at least 100. In one embodiment, the .sup.15N isotopic enrichment factor for .sup.eN is at least 200.

[0114] In one embodiment, the polypeptide comprises a sequence is at least 85% identical to SEQ ID NO: 1. In one embodiment, the polypeptide comprises a sequence is at least 90% identical to SEQ ID NO: 1. In one embodiment, the polypeptide comprises a sequence is at least 95% identical to SEQ ID NO: 1. In one embodiment, the polypeptide comprises a sequence is at least 99% identical to SEQ ID NO: 1. In one embodiment, the polypeptide comprises a sequence is identical to SEQ ID NO: 1. In one embodiment, the polypeptide comprises a sequence is at least 85% identical to SEQ ID NO: 2. In one embodiment, the polypeptide comprises a sequence is at least 90% identical to SEQ ID NO: 2. In one embodiment, the polypeptide comprises a sequence is at least 95% identical to SEQ ID NO: 2. In one embodiment, the polypeptide comprises a sequence is at least 99% identical to SEQ ID NO: 2. In one embodiment, the polypeptide comprises a sequence is identical to SEQ ID NO: 2.

[0115] In one embodiment, the SOD1 protein further comprises a post-translational modification selected from the group consisting of a phospho group, acetyl group, glutathione, crotonaldehyde, glyoxal derived carboxymethyllysine, carboxyethyl, propionyl, crotonaldehyde derived dimethyl-FDP-lysine, 4-hydroxynonenal sulfonation and nitrosylation. In one embodiment, the SOD1 protein further comprises a post-translational modification selected from the group consisting of a phospho group, acetyl group, glutathione, and crotonaldehyde.

[0116] In one aspect, provided herein is a method of preventing or treating amyotrophic lateral sclerosis in a subject, comprising (a) purifying SOD1 protein from a biological specimen from a subject to provide a purified SOD1 protein sample; (b) spiking the purified SOD1 protein sample with a defined amount of an isotopically labeled compound defined by the formula below

##STR00026##

[0117] wherein R represents hydrogen or an amine protecting group, and at least two of .sup.aC, .sup.bC, .sup.cC, .sup.dC, .sup.eN, .sup.fN, .sup.gO, and .sup.hO are isotopically labeled with a stable isotope, to provide a BMAA-spiked sample; (c) analyzing the BMAA-spiked sample by mass spectrometry; (d) measuring BMAA levels in the SOD1 protein sample by isotope dilution analysis; (e) identifying the subject for the presence or risk of amyotrophic lateral sclerosis if the BMAA levels in the SOD1 protein is greater than a normal reference value; and (f) administering to the subject L-serine in an amount sufficient to prevent or treat the amyotrophic lateral sclerosis.

[0118] In one embodiment, at least three of .sup.aC, .sup.bC, .sup.cC, .sup.dC, .sup.eN, .sup.fN, .sup.gO, and .sup.hO are isotopically labeled with a stable isotope. In one embodiment, at least four of .sup.aC, .sup.bC, .sup.cC, .sup.dC, .sup.eN, .sup.fN, .sup.gO, and .sup.hO are isotopically labeled with a stable isotope. In one embodiment, at least three of .sup.aC, .sup.bC, .sup.cC, .sup.dC, .sup.eN, and .sup.fN are isotopically labeled with a stable isotope. In one embodiment, at least four of .sup.aC, .sup.bC, .sup.cC, .sup.dC, .sup.eN, and .sup.fN are isotopically labeled with a stable isotope. In one embodiment, .sup.aC, .sup.bC, .sup.cC, .sup.dC, .sup.eN, and .sup.fN are isotopically labeled with a stable isotope. In one embodiment, the isotopically labeled compound is defined by the formula below

##STR00027##

[0119] wherein R represents hydrogen or an amine protecting group; wherein the .sup.13C isotopic enrichment factor for .sup.aC, .sup.bC, .sup.cC, and .sup.dC is at least 25; and wherein the .sup.15N isotopic enrichment factor for .sup.eN and .sup.fN is at least 100. In one embodiment, the .sup.13C isotopic enrichment factor for .sup.aC, .sup.bC, .sup.cC, and .sup.dC is at least 80 and the .sup.15N isotopic enrichment factor for .sup.eN and .sup.fN is at least 200. In one embodiment, R is hydrogen. In one embodiment, R represents a 9-fluorenylmethyloxycarbonyl group.

[0120] In one embodiment, the SOD1 protein further comprises a post-translational modification selected from the group consisting of a phospho group, acetyl group, glutathione, crotonaldehyde, glyoxal derived carboxymethyllysine, carboxyethyl, propionyl, crotonaldehyde derived dimethyl-FDP-lysine, 4-hydroxynonenal sulfonation and nitrosylation. In one embodiment, the SOD1 protein further comprises a post-translational modification selected from the group consisting of a phospho group, acetyl group, glutathione, and crotonaldehyde.

[0121] In one aspect, provided herein is a method of preventing or treating amyotrophic lateral sclerosis in a subject, comprising (a) purifying SOD1 protein from a biological specimen from a subject to provide a purified SOD1 protein sample; (b) spiking the purified SOD1 protein sample with a defined amount of a polypeptide, wherein the polypeptide includes one or more isotopically labeled .beta.-N-methylamino-L-alanine (BMAA) residues, wherein each of the one or more isotopically labeled BMAA residues is isotopically labeled with one or more stable isotopes, to provide a BMAA-spiked sample; (c) analyzing the BMAA-spiked sample by mass spectrometry; (d) measuring BMAA levels in the SOD1 protein sample by isotope dilution analysis; (e) identifying the subject for the presence or risk of amyotrophic lateral sclerosis if the BMAA levels in the SOD1 protein is greater than a normal reference value; and (f) administering to the subject L-serine in an amount sufficient to prevent or treat the amyotrophic lateral sclerosis.

[0122] In one embodiment, each of the one or more isotopically labeled BMAA residues is isotopically labeled with two or more stable isotopes. In one embodiment, each of the one or more isotopically labeled BMAA residues is defined by the formula below

##STR00028##

[0123] wherein the .sup.13C isotopic enrichment factor for .sup.dC is at least 25; and wherein the .sup.15N isotopic enrichment factor for .sup.fN is at least 100. In one embodiment, at least 80 and the .sup.15N isotopic enrichment factor for .sup.fN is at least 200. In one embodiment, the .sup.13C isotopic enrichment factor for .sup.aC, .sup.bC, and .sup.cC is at least 25. In one embodiment, the .sup.13C isotopic enrichment factor for .sup.aC, .sup.bC, and .sup.cC is at least 80. In one embodiment, the .sup.15N isotopic enrichment factor for .sup.eN is at least 100. In one embodiment, the .sup.15N isotopic enrichment factor for .sup.eN is at least 200. In one embodiment, the polypeptide comprises a sequence is at least 85% identical to SEQ ID NO: 1. In one embodiment, the polypeptide comprises a sequence is at least 90% identical to SEQ ID NO: 1. In one embodiment, the polypeptide comprises a sequence is at least 95% identical to SEQ ID NO: 1. In one embodiment, the polypeptide comprises a sequence is at least 99% identical to SEQ ID NO: 1. In one embodiment, the polypeptide comprises a sequence is identical to SEQ ID NO: 1. In one embodiment, the polypeptide comprises a sequence is at least 85% identical to SEQ ID NO: 2. In one embodiment, the polypeptide comprises a sequence is at least 90% identical to SEQ ID NO: 2. In one embodiment, the polypeptide comprises a sequence is at least 95% identical to SEQ ID NO: 2. In one embodiment, the polypeptide comprises a sequence is at least 99% identical to SEQ ID NO: 2. In one embodiment, the polypeptide comprises a sequence is identical to SEQ ID NO: 2.

[0124] In one embodiment, the SOD1 protein further comprises a post-translational modification selected from the group consisting of a phospho group, acetyl group, glutathione, crotonaldehyde, glyoxal derived carboxymethyllysine, carboxyethyl, propionyl, crotonaldehyde derived dimethyl-FDP-lysine, 4-hydroxynonenal sulfonation and nitrosylation. In one embodiment, the SOD1 protein further comprises a post-translational modification selected from the group consisting of a phospho group, acetyl group, glutathione, and crotonaldehyde.

[0125] In some embodiments, after the detection of BMAA in SOD1, the patient may be treated with L-serine. In some embodiments, after the detection of BMAA in SOD1, the patient may be treated with L-serine, or a precursor, derivative or conjugate of L-serine.

[0126] While there is no known cure for ALS, the FDA approved the use of riluzole, an anti-glutamate agent, to help slow down the progression of the disease (See U.S. Pat. No. 4,826,860). Riluzole is believed to reduce damage to motor neurons by decreasing the release of glutamate. Clinical trials with ALS patients showed that riluzole prolongs survival by several months, mainly in those with difficulty swallowing. The drug can also extend the time before an individual needs ventilation support. Other anti-glutamate agents, such as Talampanel and Memantine, have also been proposed as potential treatments for ALS. Currently, physicians can also prescribe medications to help reduce symptoms of ALS, such as fatigue, ease muscle cramps, control spasticity, and reduce excess saliva and phlegm. In some embodiments, after the detection of BMAA in SOD1, the patient may be treated with riluzole. In some embodiments, after the detection of BMAA in SOD1, the patient may be treated with talampanel or memantine.

[0127] Recently, it has been proposed to treat ALS with stem cells (See U.S. Pat. Nos. 5,968,829 and 8,765,119). It is thought that stem cells injected into the spinal cord of patients with ALS can not only mature into new nerve and spinal cells, but also release chemicals to protect existing nerve cells and their connections. Other proposed treatments include anti-apoptosis agents, such as Minocyclin, and anti-oxidative agents, such as Tamoxifen. Minocycline (see U.S. Patent App. Pub. No. 20150190415) is currently in a large Phase III clinical trial. In some embodiments, after the detection of BMAA in SOD1, the patient may be treated with stem cell therapy. In some embodiments, after the detection of BMAA in SOD1, the patient may be treated with tamoxifen or minocycline.

[0128] In one embodiment, the ALS is sporadic ALS. In one embodiment, the ALS is familial ALS.

[0129] The SOD1 protein, the BMAA-spiked sample, or a combination thereof can be prepared for analysis by mass spectrometry by a method comprising chemical reactions with flight enhancers, chemical fragmentation, enzymatic digestion, purification, or a combination thereof. In some embodiments, the isotopically labeled compounds described herein can be used to detect and quantify BMAA obtained from the hydrolytic cleavage of amino acids from a target protein as well as for identification and quantification of BMAA incorporated into proteins at the peptide and protein level utilizing isotope dilution mass spectrometry.

[0130] The proteins and peptides described herein can be used as diagnostic markers, to monitor exposure to BMAA, and/or to identify disease relevant and functionally important proteins in which BMAA has been incorporated in specific sequence locations. The proteins and peptides described herein can be utilized in a protein-cleavage-isotope dilution workflow to confirm the primary structure, and to accurately and precisely quantify BMAA incorporated into peptides produced via chemical or enzymatic digestion of specific proteins.

[0131] These proteins and peptides can also be employed in separation schemes, followed by intact mass spectrometry or peptide level detection and quantification through chemical or proteolytic digestion. These proteins and peptides can also be used to produce antibodies, aptamers and/or other affinity reagents which can be utilized for other diagnostic tools and applications. These proteins and peptides can also be used in biophysical studies, providing a method for studying the effect of this non-protein amino acid incorporation in proteins. In some embodiments, antibodies can be produced that have affinity for the SOD1 protein, or fragment of the SOD1 protein, containing a BMAA residue. In one embodiment, the BMAA residue is substituted at the Ser107 position in SOD1. In one embodiment, antibodies can be produced that have affinity for the SOD1 protein, or fragment of the SOD1 protein, containing a BMAA residue, wherein the antibodies do not have significant affinity for the corresponding SOD1 protein lacking the BMAA residue. In some embodiments, the antibody recognizes an epitope of SOD1 comprising BMAA at the Ser107 position. In some embodiments, the antibody recognizes an epitope of SOD1 that does not comprise BMAA at the Ser107 position.

[0132] The methods herein can also be used to detect .beta.-N-methylamino-L-alanine (BMAA) in additional proteins in a patient sample. In one aspect, provided herein is a method for detecting .beta.-N-methylamino-L-alanine (BMAA) in a protein, comprising (a) purifying the protein from a biological specimen to provide a purified protein sample, (b) spiking the purified protein sample with a defined amount of an isotopically labeled compound defined by the formula below

##STR00029##

[0133] wherein R represents hydrogen or an amine protecting group, and at least two of .sup.aC, .sup.bC, .sup.cC, .sup.dC, .sup.eN, .sup.gO, and .sup.hO are isotopically labeled with a stable isotope, to provide a BMAA-spiked sample; (c) analyzing the BMAA-spiked sample by mass spectrometry; and (d) measuring BMAA levels in the protein sample by isotope dilution analysis.

[0134] In one aspect, provided herein is a method for detecting .beta.-N-methylamino-L-alanine (BMAA) in a protein, comprising: (a) purifying the protein from a biological specimen to provide a purified protein sample; (b) spiking the purified protein sample with a defined amount of a polypeptide, wherein the polypeptide includes one or more isotopically labeled .beta.-N-methylamino-L-alanine (BMAA) residues, wherein each of the one or more isotopically labeled BMAA residues is isotopically labeled with one or more stable isotopes; to provide a BMAA-spiked sample; (c) analyzing the BMAA-spiked sample by mass spectrometry; and (d) measuring BMAA levels in the protein sample by isotope dilution analysis.

[0135] In another aspect, provided herein is a method of detecting or predicting amyotrophic lateral sclerosis in a subject, comprising (a) purifying a protein from a biological specimen from a subject to provide a purified protein sample; (b) spiking the purified protein sample with a defined amount of an isotopically labeled compound defined by the formula below

##STR00030##

[0136] wherein R represents hydrogen or an amine protecting group, and at least two of .sup.aC, .sup.bC, .sup.cC, .sup.dC, .sup.eN, .sup.fN, .sup.gO, and .sup.hO are isotopically labeled with a stable isotope, to provide a BMAA-spiked sample; (c) analyzing the BMAA-spiked sample by mass spectrometry; (d) measuring BMAA level in the protein sample by isotope dilution analysis; and (e) identifying the subject for the presence or risk of amyotrophic lateral sclerosis if the BMAA level in the protein is greater than a normal reference value.

[0137] In one aspect, provided herein is a method of preventing or treating amyotrophic lateral sclerosis in a subject, comprising (a) purifying a protein from a biological specimen from a subject to provide a purified protein sample; (b) spiking the purified protein sample with a defined amount of an isotopically labeled compound defined by the formula below

##STR00031##

[0138] wherein R represents hydrogen or an amine protecting group, and at least two of .sup.aC, .sup.bC, .sup.cC, .sup.dC, .sup.eN, .sup.fN, .sup.gO, and .sup.hO are isotopically labeled with a stable isotope, to provide a BMAA-spiked sample; (c) analyzing the BMAA-spiked sample by mass spectrometry; (d) measuring BMAA levels in the protein sample by isotope dilution analysis; (e) identifying the subject for the presence or risk of amyotrophic lateral sclerosis if the BMAA levels in the protein is greater than a normal reference value; and (f) administering to the subject L-serine in an amount sufficient to prevent or treat the amyotrophic lateral sclerosis.

[0139] In still a further aspect, provided herein is a method of preventing or treating amyotrophic lateral sclerosis in a subject, comprising (a) purifying a protein from a biological specimen from a subject to provide a purified protein sample; (b) spiking the purified protein sample with a defined amount of a polypeptide, wherein the polypeptide includes one or more isotopically labeled .beta.-N-methylamino-L-alanine (BMAA) residues, wherein each of the one or more isotopically labeled BMAA residues is isotopically labeled with one or more stable isotopes, to provide a BMAA-spiked sample; (c) analyzing the BMAA-spiked sample by mass spectrometry; (d) measuring BMAA levels in the protein sample by isotope dilution analysis; (e) identifying the subject for the presence or risk of amyotrophic lateral sclerosis if the BMAA levels in the protein is greater than a normal reference value; and (f) administering to the subject L-serine in an amount sufficient to prevent or treat the amyotrophic lateral sclerosis.

[0140] The proteins or polypeptides used in the methods described herein can comprise any of the proteins or protein fragments described in Table 6 or Table 7 below.

[0141] By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below.

EXAMPLES

Example 1

Preparation of .sup.13C.sub.4.sup.15N.sub.2 .beta.-N-Methylamino-L-Alanine

[0142] Preparation of Compound 1. To a solution of .sup.13C-.sup.15N labeled L-asparagine monohydrate (95.8 mg, 0.614 mmol) in 10% aqueous Na.sub.2CO.sub.3 (1.6 mL) was added 1,4-dioxane (0.9 mL) and the mixture was cooled to 0.degree. C. Benzyl chloroformate (130 mg, 0.737 mmol) was then added and the mixture was allowed to warm to rt overnight. The reaction mixture was poured into water (4.0 mL), and the mixture was extracted with diethyl ether (.times.3). The aqueous layer was then acidified with an aqueous solution of 2N HCl (pH=2), and the white solid was filtered to afford 98.5 mg (59%) of the product: .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.37 (m, 5H), 5.10 (m, 2H), 4.25 (bd, 1H, J=132.1 Hz), 2.81 (bd, 1H, J=44.1 Hz), 2.46 (bd, 1H, J=64.6 Hz); ESIMS m/z 273 [M+H].sup.+; HRMS m/z calculated for .sup.13C.sub.4C.sub.8H.sub.14.sup.15N.sub.2O.sub.5 [M+Na].sup.+ 295.0870, found 295. 0867.

##STR00032##

[0143] Preparation of Compound 2. To a slurry of N.sup.2-benzyloxycarbonylasparagine (98.5 mg, 0.362 mmol) in ethyl acetate (0.89 mL), acetonitrile (0.96 mL), and water (0.46 mL) was added iodosobenzene diacetate (0.166 g, 0.507 mmol) at 15.degree. C. and the mixture was stirred for 30 min at 15.degree. C. The reaction mixture was then allowed to warm to rt and stirred until completion (4 h). The mixture was cooled to 5.degree. C., and the product was collected, washed with ethyl acetate, and dried in vacuo to afford 33.9 mg (39%) of the product as a white solid: ESIMS m/z 244 [M+H].sup.+. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.03 (brs, 1H), 7.85 (brs, 2H), 7.40 (s, 5H), 5.07 (s, 2H), 4.30 (bd, 1H, J=139.2 Hz), 3.31 (bd, 1H, J=83.9 Hz), 3.31 (bd, 1H, J=83.9 Hz), 2.95 (bd, 1H, J=87.9 Hz); ESIMS m/z 244 [M+H].sup.+; HRMS m/z calculated for .sup.13C.sub.3C.sub.8H.sub.14.sup.15N.sub.2O.sub.4 [M+H].sup.+ 244.1068, found 244.1066.

##STR00033##

[0144] Preparation of Compound 3. To a suspension of compound 3 (33.9 mg, 0.139 mmol) in methanol (0.60 mL) was added Et.sub.3N (42.3 mg, 0.416 mmol) and benzaldehyde (29.6 mg, 0.278 mmol) at rt, and the mixture was stirred for 30 min. The reaction mixture was cooled to 0.degree. C., followed by the addition of NaBH.sub.4 (16.0 mg, 0.416 mmol). The mixture was then stirred for an additional 15 min at 0.degree. C., and concentrated under reduced pressure. The residue was then dissolved in 0.1 M aqueous solution of NaOH, and extracted with diethyl ether (.times.3). The aqueous layer was then acidified with an aqueous solution of 10% hydrochloric acid, and the resultant white precipitate was filtered to afford 27.2 mg of the product. The white solid was dissolved in methanol (0.27 mL), and a solution of 35% aqueous solution of formaldehyde (18.2 .mu.L, 0.244 mmol) was added. The reaction was stirred for an additional 15 min, and cooled to 0.degree. C. NaBH.sub.4 (9.32 mg, 0.244 mmol) was then added, and the mixture was stirred for 15 min. Upon completion, the mixture was concentrated under reduced pressure, and the crude residue was dissolved in water, acidified (pH=6) with a 1 M aqueous solution of HCl, extracted with CHCl.sub.3, dried (MgSO.sub.4), and concentrated under reduced pressure to afford the crude product. The crude product was triturated with diethyl ether to afford 28.3 mg (100%) of the product as a white solid: .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.18 (s, 10H), 6.38 (d, 1H, J=92.5 Hz), 5.09 (d, 1H, J=16.5 Hz), 4.88 (d, 1H, J=15.4 Hz), 4.19 (d, 1H, J=83.9 Hz), 3.58 (d, 1H, J=13.2 Hz), 3.48 (d, 1H, J=16.1 Hz), 3.01 (s, 1H), 2.67 (s, 1H), 2.12 (s, 3H); ESIMS m/z 348 [M+H].sup.+; HRMS m/z calculated for .sup.13C.sub.3C.sub.16H.sub.22.sup.15N.sub.2O.sub.4 [M+H].sup.+ 348.1694, found 348.1690.

##STR00034##

[0145] Preparation of Compound .sup.13C.sub.4.sup.15N.sub.2 .beta.-N-Methylamino-L-Alanine. To a degassed solution of compound 3 (28.3 mg, 0.0814 mmol) in methanol (0.8 mL) was added Pd/C (8.66 mg, 0.00813 mmol), and the mixture was further degassed for an additional 5 min. The mixture was then saturated with H.sub.2 gas and stirred under a H.sub.2 atmosphere overnight. The Pd/C was filtered through Celite.RTM., and washed with methanol. The filtrated was concentrated under reduced pressure and the crude product was triturated with diethyl ether to afford 7.7 mg (77%) of the product as a white solid: ESIMS m/z 146 [M+Na].sup.+; HRMS m/z calculated for .sup.13C.sub.3CH.sub.10.sup.15N.sub.2O.sub.2 [M+H].sup.+ 124.0856, found 124.0856. This final product was further characterized by MS/MS to confirm the exact structure (FIG. 14).

##STR00035##

.sup.13C.sub.4.sup.15N.sub.2 .beta.-N-methylamino-L-alanine

[0146] A well-characterized, highly phosphorylated protein, Beta-Casein, was utilized as a positive control. The intact protein was reacted with methylamine under the same conditions for peptide synthesis, converting phosphoserines to BMAA. Purified SOD1 from 3 patients with sporadic ALS and 3 healthy controls were washed on a 10 kDa FASP filter (Millipore) and concentrated to 50 .mu.L. 20 .mu.g of protein was hydrolyzed using 50 of 6N HCl and incubating at 110.degree. C. for 18 hours. 5 .mu.L of 60.9 mM SIL BMAA was spiked into the samples post-hydrolysis. Samples were then dried and resuspended in 100 .mu.L of 0.001% Zwittergent 3-16. Direct infusion ESI MS/MS of SIL BMAA confirmed the location of isotope incorporation. A ZipChip (908 Devices) capillary electrophoresis was utilized to separate BMAA prior to electrospray ionization mass spectrometry. Accurate intact mass and migration time of the SIL reagent was used to identify BMAA.

[0147] The peptide sequence DGVADVSIEDSVISLSGDHCIIGR (SEQ ID NO:17) with highlighted (bold and underlined) Serine phosphorylated, carbamidomethylated Cysteine and .sup.13C.sub.6.sup.15N.sub.4 isotopically labeled Arginine was obtained from New England Peptide. This sequence was validated by accurate intact mass (FIG. 2) and MS/MS (FIG. 3) which allowed for site specific confirmation of phosphorylation. A solution of water, DMSO, EtOH solution in 2:2:1 mixture containing 0.1M Barium Hydroxide and 1M Methylamine pH 12.5 was used for derivatization of the SIL peptide. 5 (5 .mu.g) of peptide was added to 20 .mu.L of derivatization solution and incubated at 37.degree. C. for two hours. The reaction was quenched with 2 .mu.L of Acetic Acid. See FIG. 1 for synthesis reference. The formation of BMAA at the phosphorylated Serine residue was confirmed by accurate intact mass (FIG. 2) and MS/MS (FIG. 4). Further, the R.T of the BMAA peptide was distinctly different from that of the phosphorylated peptide (FIG. 5), along with co-elution of fragment ions to confirm the identity of this species. The conserved relative abundance of the fragments belonging to the SIL BMAA peptide act as a standard for further confirmation.

[0148] Each sample was diluted 2-fold in a denaturing solution of 100 mM DTT (15.43 mg/mL) and 8 M urea. Then the samples were incubated at 56.degree. C. for 30 minutes. After incubation enough alkylation solution, made from 1 M iodoacetamide (184.96 mg/mL) and 8 M urea, was added to give each sample a final iodoacetamide concentration of 200 mM. Then the samples were incubated at 37.degree. C. for an hour. The appropriate amount for each sample was pipetted into an Amicon Ultra-0.1 MWCO-filter unit (10 kDa, Millipore). These were centrifuged for 15 minutes at 14,000.times.g and 20.degree. C.; when finished the eluent was discarded. The remaining volume was diluted with 400 uL of a digestion buffer made of 2 M urea (102.12 mg/mL) and 10 mM CaCl_2 (1.11 mg/mL). The samples were centrifuged again for 15 minutes at 14,000.times.g and 20.degree. C. This was repeated two more times, making sure to discard the eluent after each run. After the final centrifugation the collection tubes were changed and 45 uL of modified porcine trypsin reconstituted in 2 M urea and 10 mM CaCl_2 was added and the samples were incubated at 37.degree. C. overnight. Then the samples were quenched with 50 uL of 1% formic acid (v/v) and 0.001% Zwittergent 3-16 and centrifuged for 15 minutes at 14,000.times.g and 20.degree. C. 400 uL of quench buffer was added to the retained volume and centrifuged again for 15 minutes at 14,000.times.g and 20.degree. C. Samples were frozen at -80.degree. C. and lyosphilized in a speedvac. Immediately prior to analysis samples were reconstituted in 90 uL of Zwittergent 3-16. A BMAA peptide dry aliquot of 5 ug was resuspended in 500 uL of Zwittergent. A 45 uL aliquot of the protein digest was then spiked with 5 uL of BMAA peptide standard for LC-MS analysis.

[0149] LC-MS/MS by parallel reaction monitoring was used to isolate, fragment and perform accurate mass measurements of our target endogenous BMAA peptide and SIL BMAA peptides. Direct inject column configuration on a Thermo Easy nano-LC 1000 system coupled to a QExactive High Field mass spectrometer was used. Analytical columns were made using 75 um.times.15 cm PicoFrit columns (New Objective, Woburn, Mass.) which were self-packed with Kinetex C18 2.6-um particles (phenomenex, Torrance, Calif., USA). The samples were loaded with a 10 uL injection volume of mobile phase A (98% water, 2% acetonitrile, and 0.2% formic acid) with a max pressure of 500 Bar. A 45 minute run from 2% to 30% mobile phase B (98% acetonitrile, 2% water, and 0.2% formic acid) was performed at a flow rate of 300 nL/min. The analysis had the following parameters: a spray voltage of +1750.00, capillary temperature of 325.degree. C., a S-lens RF level of 65.00, a MS/MS resolving power of 15,000, a 1e6 AGC target, a 1,000 ms fill time, a 2.5 m/z isolation window with an isolation offset of 1.0 m/z, a fixed first mass of 125.0 m/z, and a 20, 30 stepped normalized collision energy. There was an inclusion list containing 846.4228 m/z and 843.0867 m/z.

[0150] Without the SIL BMAA peptide, correct identification of this peak becomes very difficult as other mass conflicts are present within 5 ppm of the endogenous BMAA peptide as shown in FIG. 7 where the most abundant peak is the same sequence does not co-elute with the SIL peptide. When examining the correct retention time, the intact mass appears to be present within 1.2 ppm mass accuracy and the correct charge state (+3) is also identified (FIG. 8). Moreover, this peak co-elutes with our SIL peptide as shown in FIG. 9. Fragment ions corresponding to the endogenous peptide with BMAA at the expected location could be identified at the same retention time (FIG. 13).

Example 2

NanoLC MS and MS/MS Characterization of SOD1 in Human Clinical ALS

[0151] Samples for analysis were obtained from control patients (n=10) and ALS patients (n=7). The SOD1 protein was purified from plasma in each of these patient samples. The SOD1 protein can be purified by one of skill in the art (for example, purification using an antibody directed to SOD1 or using a protein that has binding affinity for SOD1). In some experiments, the intact SOD1 protein was analyzed, while in other experiments, the SOD1 protein was digested with trypsin before further analysis (See FIG. 10).

[0152] For the liquid chromatography experiments, the Thermo Easy nanoLC setup included: 75 .mu.m inner diameter 15 cm column utilizing C18 stationary phase for reversed-phase separation of peptides base on hydrophobicity. The flow rate was 300 nL/min. 2 .mu.L of digested (trypsin) SOD1 containing .about.200 ng was injected directly onto the column using a Mobile phase A (98% water, 2% Acetonitrile (ACN), 0.2% formic acid) and a Mobile phase B (98% ACN, 2% water, 0.2% formic acid). A gradient elution was performed from 5%-40%B over 30 min.

[0153] Data was collected by Full-MS data dependent MS/MS using a top 20 experiment on Q Exactive HF.

[0154] For intact protein analysis, 200 ng of undigested protein was injected directly on a 10 cm column using an isocratic (50% A/50% B) elution. Accurate Mass can be obtained by deconvolution with Xtract algorithm.

[0155] For the initial SOD1 proteomics search parameters, 69 protein sequences including SOD1 wild-type sequence as well as commonly found sample contaminants such as keratin and trypsin were identified in protein databases. Protein DB is digested in silico using rules for tryptic digestion (Cleaves C-terminal to R/K except when preceded by P) and allowing for potential missed cleavages (up to 3). The search tolerance allowed for 5 ppm MMA (mass measurement accuracy) for peptide, 0.02 Da for fragment ions. There were two bioinformatics workflows allowing for:

[0156] 1) Dynamic Modifications: Ser.fwdarw.BMAA, Glutathionylation (Cys), Deamidation (Q,N), Phospho (S,T,Y), Carbamidomethylation (Cys). Carboxyethyl (MDA), Crotonaldehyde (C,H), 4-HNE (K), Acrolein/proprionyl (K, T, S), Acetylation (K), Nitrotryptophan (W), Sulfonylation (C, T, S), Glyoxal derived carboxymethyllysine CML (K), Crotonaldehyde derived dimethyl-FDP-lysine (K)

[0157] 2) SNP Dynamic Modifications: Val.fwdarw.Met, Ala.fwdarw.Val, His.fwdarw.Arg, Thr.fwdarw.Arg, Gly.fwdarw.Arg, Ser.fwdarw.BMAA (Also searched all other amino acids to BMAA)

[0158] Fixed: Carbamidomethylation

[0159] Peptides were filtered at 1% False Discovery Rate (FDR).

[0160] The total glutathionylation and phosphorylation from 17 patient samples (10 healthy controls and 7 ALS patient samples) were analyzed and results are shown in Table 1.

TABLE-US-00004 TABLE 1 Glutathionylation + Sample Glutathionylation Phosphorylation Phosphorylation HC01 36.31% 14.53% 6.19% HC02 39.17% 21.69% 10.98% HC03 30.22% 19.16% 5.39% HC04 33.16% 20.67% 6.10% HC05 28.06% 20.38% 5.19% HC06 34.77% 20.82% 6.54% HC07 41.66% 11.35% 5.12% HC08 35.73% 12.66% 3.85% HC09 19.35% 12.97% 1.96% HC10 34.94% 14.09% 4.94% ALS01 28.26% 33.91% 9.01% ALS02 0.00% 79.15% 0.00% ALS03 2.34% 72.06% 0.00% ALS04 27.75% 24.76% 4.58% ALS06 38.99% 22.18% 8.09% ALS08 38.84% 19.88% 7.32% ALS09 43.98% 20.00% 11.27% p value 0.23 0.02 0.94 *HC = Healthy Control sample *ALS = Amyotrophic Lateral Sclerosis sample

[0161] The post-translational modifications identified from the 17 patient samples were analyzed and results are shown in Table 2.

TABLE-US-00005 TABLE 2 HC MOD ALS MOD Shared Occupancy C111(Glutathione) T2(Phospho) K128(Acetyl) K23(Acetyl) K136(Acetyl) S68(Phospho) K3(Acetyl) K75(Acetyl) K9(Acetyl) T78(Phospho) T39(Phospho) 0.85% HC, 0.89% UNC, p = 0.86 S107(Phospho) K70(Acetyl) C146(Glutathione) T88(Phospho) 0.44% HC, 0.42% UNC, p = 0.94 K91(Acetyl) K122(Acetyl) T54(Phospho) 51% HC, 45% UNC, p = 0.84 T58(Phospho) K30(Acetyl) *HC = Healthy Control sample *ALS = Amyotrophic Lateral Sclerosis sample *The amino acid numbering shown in the table does not include the starting methionine.

[0162] Known modifications in Table 2 include: C111(Glutathione), S107(Phospho), T2(Phospho), K3(Acetyl), and K122(Acetyl). The other modifications identified in Table 2 are novel modifications that have not previously been identified. Modifications identified with high confidence are shown in FIG. 11, and summarized in Table 3.

TABLE-US-00006 TABLE 3 Amino Acid Modification Peptide T2 phosphorylation ATKAVCVLKGDGPVQGIINFEQK (SEQ ID NO: 7) K3 acetylation ATKAVCVLKGDGPVQGIINFEQK (SEQ ID NO: 7) C6 sulfo AVCVLKGDGPVQGIINFEQK (SEQ ID NO: 8) K9 CML AVCVLKGDGPVQGIINFEQKESNGPVKVWGSIK (SEQ ID NO: 9) K9 acetylation ADDLGKGGNEESTK (SEQ ID NO: 10) K23 CML AVCVLKGDGPVQGIINFEQKESNGPVKVWGSIK (SEQ ID NO: 9) K30 propionyl ESNGPVKVWGSIK (SEQ ID NO: 11) W32 nitro ESNGPVKVWGSIK (SEQ ID NO: 11) T39 phosphorylation GLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR (SEQ ID NO:12) T54 sulfo GLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR (SEQ ID NO: 12) T54 propionyl GLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR (SEQ ID NO: 12) C57 crotonaldehyde GLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR (SEQ ID NO: 12) T58 sulfo GLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR (SEQ ID NO: 12) T88 phosphorylation HVGDLGNVTADK (SEQ ID NO: 13) K91 crotonaldehyde HVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGR (SEQ derived dimethyl- ID NO: 14) FDP-lysine K91 CML DEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGR (SEQ ID NO: 15) S102/ phosphorylation DFVADVSIEDSVISLSGDHCIIGRTLVVHEK (SEQ ID 105 NO: 16) S107 phosphorylation HVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGR (SEQ ID NO: 14) S107 Ser.fwdarw.BMAA DGVADVSIEDSVISLSGDHCIIGR (SEQ ID NO: 17) H110 crotonaldehyde DGVADVSIEDSVISLSGDHCIIGR (SEQ ID NO: 17) C111 glutathione HVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGR (SEQ ID NO: 14) C111 crotonaldehyde DGVADVSIEDSVISLSGDHCIIGR (SEQ ID NO: 17) T116 propionyl TLVVHEKADDLGK (SEQ ID NO: 18) K122 CML TLVVHEKADDLGKGGNEESTKTGNAGSR (SEQ ID NO: 19) K122 propionyl TLVVHEKADDLGK (SEQ ID NO: 18) K122 acetylation TLVVHEKADDLGK (SEQ ID NO: 18) K128 carboxyethyl ADDLGKGGNEESTK (SEQ ID NO: 10) K128 propionyl ADDLGKGGNEESTK (SEQ ID NO: 10) G130 Gly.fwdarw.Arg ADDLGKGGNEESTKTGNAGSR (SEQ ID NO: 19) K136 propionyl ADDLGKGGNEESTKTGNAGSR (SEQ ID NO: 19) K136 acetylation GGNEESTKTGNASGSR (SEQ ID NO: 20) C146 glutathione TGNAGSRLACGVIGIAQ (SEQ ID NO: 21) *The amino acid numbering shown in the table does not include the starting methionine.

[0163] Modifications identified with medium confidence are shown in FIG. 12 and summarized in Table 4.

TABLE-US-00007 TABLE 4 Amino Acid Modification Peptide S25 propionyl ESNGPVKVWGSIK (SEQ ID NO: 11) K36 CML VWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR (SEQ ID NO: 22) T39 propionyl GLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR (SEQ ID NO: 12) H46 carboxyethyl GLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR (SEQ ID NO: 12) H48 carboxyethyl GLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR (SEQ ID NO: 12) A55 HNE VWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR (SEQ ID NO: 22) C57 sulfo GLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR (SEQ ID NO: 12) C57 HNE GLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR (SEQ ID NO: 12) T58 propionyl GLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR (SEQ ID NO: 12) S59 sulfo GLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR (SEQ ID NO: 12) S59 propionyl GLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR (SEQ ID NO: 12) H80 carboxyethyl HVGDLGNVTADK (SEQ ID NO: 13) K91 HNE HVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGR (SEQ ID NO: 14) K91 acetylation HVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGR (SEQ ID NO: 14) A95 HNE HVGKLGNVTADKDGVADVSIEDSVISLSGDHCIIGR (SEQ ID NO: 23) S105 propionyl HVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGR (SEQ ID NO: 14) S107 propionyl HVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGR (SEQ ID NO: 14) *The amino acid numbering shown in the table does not include the starting methionine.

[0164] The single nucleotide polymorphisms (SNPs) identified from the 17 patient samples were analyzed and results are shown in Table 5.

TABLE-US-00008 TABLE 5 HC Sequence Variant ALS Sequence Variant Shared Sequence Variant V5(Val->Met) G10(Gly->Arg) G130(Gly->Arg) V103(Val->Met) G12(Gly->Arg) V31(Val->Met) G44(Gly->Arg) G27(Gly->Arg) V47(Val->Met) G51(Gly->Arg) G82(Gly->Arg) G72(Gly->Arg) G127(Gly->Arg) G16(Gly->Arg) * The amino acid numbering shown in the table does not include the starting methionine.

Known single nucleotide polymorphisms (SNPs) in Table 5 include: G12(Gly.fwdarw.Arg). The additional SNPs identified in Table 5 are novel.

[0165] Using the novel reagents and methods disclosed herein, the inventors have identified the incorporation of endogenous BMAA into the SOD1 protein in human ALS samples for the first time. Incorporation of BMAA at Serine 107 may act alone, or in combination with other BMAA substitutions, SNP(s) and/or post translational modifications (PTMs) on SOD1, other SOD proteins, or in combination with SOD1 binding partners. Additionally, other proteins within the oxidative stress pathway could incorporate this non-natural amino acid. In the case of SOD1, incorporation of this non-natural amino acid could also reflect tRNA synthetase mutations or promiscuity.

[0166] In some embodiments, the consequences of BMAA incorporation could include, for example, a decrease in the stability of the homodimer, an increase in the likelihood of SOD1 to aggregate (e.g., trimers which are very toxic), a decrease in the ability of SOD1 to bind metals, SOD1 misfolding, a decrease or alteration of enzymatic activity, and/or tRNA synthetases have sequence variation altering their specificity.

Example 3

NanoLC MS and MS/MS Characterization of BMAA Incorporation Sites in Human Clinical ALS Brain Tissue

[0167] Brain tissue samples of 12 matched ALS/healthy control samples were obtained from Emory University. 100.times. Halt protease/phosphatase inhibitor was diluted to 1.times. in a lysis buffer which comprised 8 M urea and 100 mM ammonium bicarbonate at a pH of 7.0. Samples were blended in a Bullet Blender with 500 .mu.L of lysis buffer and 750 .mu.g of stainless steel beads for 15 minutes at a setting of 3 while refrigerated. A Dismembranator Sonicator was used three times for 5 seconds with 15 second breaks. Each sample was diluted 4-fold with water such that the concentration of Urea was 2M. The protein concentration of each sample was then determined using a Bradford Assay. An appropriate volume of the sample containing 250 .mu.g was transferred to a 10 kDa MWCO-filter unit and diluted 2-fold in a denaturing solution made from 100 mM DTT and 8 M urea and heated at 56.degree. C. for 30 minutes. The samples were alkylated using 1 M iodoacetamide and 8 M urea to give a final iodoacetamide concentration of 200 mM and heated at 37.degree. C. for 60 minutes. To concentrate the samples, they were spun for 15 minutes at 14,000.times.g and 20.degree. C. The retained volume was diluted three times with 400 .mu.L of a digesting buffer of 100 mM ammonium bicarbonate at pH 7.0 and spun for 15 minutes at 14,000.times.g and 20.degree. C. between each buffer addition. Filters were then transferred to a clean tube. 2 .mu.g SIL SOD1 surrogate peptide containing BMAA was reconstituted in 500 .mu.L of 0.001% Zwittergent 3-16, then 5 .mu.L was spiked into each sample. The samples were digested using 45 .mu.L of an enzyme solution containing 100 .mu.g/ml modified trypsin and 100 mM ammonium bicarbonate to give a 1:50 enzyme:protein ratio and incubated overnight at 37.degree. C. 50 .mu.L of a quench buffer made from 1% formic acid and 0.001% Zwittergent 3-16 was added to the filter units which were then spun for 15 minutes at 14,000.times.g to elute the peptides. 400 .mu.L of the quench buffer was added to the retained volume and spun for 15 minutes at 14,000.times.g using the same collection tube. The samples were lyophilized and kept at -80.degree. C. until analysis. Samples were reconstituted in 100 .mu.L of 0.001% Zwittergent 3-16. 4 .mu.L (approximately 0.5 .mu.g) was injected via direct inject column configuration onto a Thermo Easy nano-LC 1200 system coupled to a QExactive High Field mass spectrometer. Analytical columns were made using 75 um.times.15 cm PicoFrit columns (New Objective, Woburn, Mass.) which were self-packed with Kinetex C18 2.6-um particles (phenomenex, Torrance, Calif., USA). The samples were loaded with a 8 uL injection volume of mobile phase A (98% water, 2% acetonitrile, and 0.2% formic acid) with a max pressure of 600 Bar. A 4 hour gradient was performed at 300 nL/min, going from 2% mobile phase B (80% Acetonitrile, 20% water, 0.2% formic acid), to 40%. A top 20 data dependent acquisition was performed with the following MS parameters: a spray voltage of +2 kV, capillary temperature of 325.degree. C., a S-lens RF level of 65.00, an MS resolving power of 120 k and an MS/MS resolving power of 15,000, a 3e6 MS AGC target with a max fill time of 50 ms and 1E5 MS/MS AGC with a max fill time of 30 ms and an intensity threshold of 3.3E4. The data was processed through proteome discoverer 2.0 and searched using the Uniprot human proteome FASTA database. Protein DB was digested in silico using rules for tryptic digestion (Cleaves C-terminal to R/K except when preceded by P) and allowing for potential missed cleavages (up to 3). The search tolerance allowed for 5 ppm MMA (mass measurement accuracy) for peptide, 0.02 Da for fragment ions. The following variable modifications were searched:

[0168] Serine.fwdarw.BMAA, Carbamidomethylation (Cys), Crotonaldehyde (C,H), and Aspartic acid.fwdarw.Glutamine

[0169] Peptides were validated at a 1% False Discovery Rate and manually validated to ensure high sequence coverage and exclude alternative hypotheses (Aspartic acid 4 Glutamine or Crotonaldehyde Cys/His+Carbamidomethylation). All peptides containing a Ser.fwdarw.BMAA which could be validated by site specific mass fragmentation are listed below in Table 6, and the corresponding wild-type sequence fragments (without BMAA incorporation) are listed below in Table 7.

TABLE-US-00009 TABLE 6 Validated Peptides Containing BMAA Protein (Accession # other Sequence No.) Protein Control ALS mods AYHEQLSVAEITS*S**C*F Q9NY65 Human Tubulin alpha- 1 3 1 EPNSQMVK (SEQ ID NO: 24) 8 chain protein (carbami- sequence fragment domethyl) DLYANTVLSGG*S*TMYPG Q562R1 Human Beta-actin-like 0 1 0 IADR (SEQ ID NO: 25) protein 2 protein sequence fragment DTI*C*EE*S*LR (SEQ ID Q8NG31 Human Kinetochore 0 1 1 NO: 26) scaffold 1 protein (crotonal- sequence fragment dehyde) ESVSS*S*DR (SEQ ID P40145 Human Adenylate 0 1 0 NO: 27) cyclase type 8 protein sequence fragment GYECILNIQG*S*EQR (SEQ Q9HCM2 Human Plexin 4A 1 0 0 ID NO: 28) protein sequence fragment fragment HTGPGLLSMANSGPNTNG* Q9UNP9 Human Peptidyl- 4 2 0 S*QFFLTCDK (SEQ ID prolyl cis-trans NO: 29) isomerase E peptide sequence fragment KATYVYET*S*GPNLSDNK A6NE01 Human Protein 1 0 0 SGQK (SEQ ID NO: 30) FAM186A protein sequence fragment KGMW*S*EGNGSHTIR Q7KZF4 Human 0 1 0 (SEQ ID NO: 31) Staphylococcal nuclease domain- containing protein 1 protein sequence fragment KT*S*TDFSEVIK (SEQ ID Q01484 Human Ankyrin-2 0 1 0 NO: 32) protein sequence fragment LGQG*S*GQGPK (SEQ ID Q9BSW3 Human EF-hand 1 0 0 NO: 33) calcium-binding domain-containing protein 4B protein sequence fragment LLLGT*S*GEGK (SEQ ID Q9Y4C4 Human Malignant 0 1 0 NO: 34) fibrous histiocytoma- amplified sequence 1 protein sequence fragment LSLMLDEG*S*SCPTPAK Q9ULV5 Human Heat shock 0 1 0 (SEQ ID NO: 35) factor protein 4 protein sequence fragment MSDILRELLCV*S*EK (SEQ P49441 Human Inositol 1 0 0 ID NO: 36) polyphosphate 1- phosphatase protein sequence fragment NTA*S*HTAAAAR (SEQ ID Q9Y666 Human Solute carrier 0 1 0 NO: 37) family 12 member 7 protein sequence fragment Q*S*ELSAEESPEK (SEQ ID Q567U6 Human Coiled-coil 1 2 0 NO: 38) domain-containing protein 93 protein sequence fragment *S**C*TAADTAAQITQR Q29960 Human HLA class I 2 1 1 (SEQ ID NO: 39) histocompatibility (crotonal- antigen, Cw-16 alpha dehyde) chain protein sequence fragment SGGGGNFVL*S*TSLVGYL Q9UKG9 Human Peroxisomal 1 0 0 R (SEQ ID NO: 40) carnitine O- octanoyltransferase protein sequence fragment *S*GTSIPSAGK (SEQ ID Q7Z5P9 Human Mucin-19 1 0 0 NO: 41) protein sequence fragment *S*LAGPAGAAPAPGLGAA Q8NES3 Human Beta-1,3-N- 1 0 0 AAAPGALVR (SEQ ID acetylglucosaminyltra NO: 42) nsferase lunatic fringe protein sequence fragment SLFPPWTFQFQ*S*GDLEEK Q8NDH2 Human coiled-coil 0 1 0 (SEQ ID NO: 43) domain-containing protein 168 protein sequence fragment SLGGAVGSVA*S*GAR Q8NHG8 Human E3 ubiquitin- 1 0 0 (SEQ ID NO: 44) protein ligase ZNRF2 protein sequence fragment *S*PVTFLSDLR (SEQ ID A5YKK6 CCR4-NOT 1 0 0 NO: 45) transcription complex subunit 1 protein sequence fragment *S*TLVHSLFLTDLYK (SEQ Q99719 Human Septin-5 3 0 0 ID NO: 46) protein sequence fragment VEELIE*S*EAPPK (SEQ ID Q96B23 Human 1 0 0 NO: 47) Uncharacterized Protein C18orf25 protein sequence fragment V*S*ELEDFINGPNNAHIQQ P53675 Human Clathrin heavy 5 3 0 VGDR (SEQ ID NO: 48) chain 2 protein sequence fragment YI*S*DLK (SEQ ID NO: 49) P55774 Human C-C motif 0 1 0 chemokine 18 protein sequence fragment *S* = Ser.fwdarw.BMAA *C* = Carbamidomethylation or Crotonaldehyde

TABLE-US-00010 TABLE 7 Wild Type Sequences for Peptides Identified in Table 6 Protein SEQ (Accession Sequence ID NO No.) Protein AYHEQLSVAEITSSCFEPNSQMVK SEQ ID Q9NY65 Human Tubulin alpha-8 NO: 50 chain protein sequence fragment DLYANTVLSGGSTMYPGIADR SEQ ID Q562R1 Human Beta-actin-like NO: 51 protein 2 protein sequence fragment DTICEESLR SEQ ID Q8NG31 Human Kinetochore NO: 52 scaffold 1 protein sequence fragment ESVSSSDR SEQ ID P40145 Human Adenylate cyclase NO: 53 type 8 protein sequence fragment GYECILNIQGSEQR SEQ ID Q9HCM2 Human Plexin 4A protein NO: 54 sequence fragment fragment HTGPGLLSMANSGPNTNGSQFFLTC SEQ ID Q9UNP9 Human Peptidyl-prolyl cis- DK NO: 55 trans isomerase E peptide sequence fragment KATYVYETSGPNLSDNKSGQK SEQ ID A6NE01 Human Protein FAM186A NO: 56 protein sequence fragment KGMWSEGNGSHTIR SEQ ID Q7KZF4 Human Staphylococcal NO: 57 nuclease domain- containing protein 1 protein sequence fragment KTSTDFSEVIK SEQ ID Q01484 Human Ankyrin-2 protein NO: 58 sequence fragment LGQGSGQGPK SEQ ID Q9BSW3 Human EF-hand calcium- NO: 59 binding domain- containing protein 4B protein sequence fragment LLLGTSGEGK SEQ ID Q9Y4C4 Human Malignant fibrous NO: 60 histiocytoma-amplified sequence 1 protein sequence fragment LSLMLDEGSSCPTPAK SEQ ID Q9ULV5 Human Heat shock factor NO: 61 protein 4 protein sequence fragment MSDILRELLCVSEK SEQ ID P49441 Human Inositol NO: 62 polyphosphate 1- phosphatase protein sequence fragment NTASHTAAAAR SEQ ID Q9Y666 Human Solute carrier NO: 63 family 12 member 7 protein sequence fragment QSELSAEESPEK SEQ ID Q567U6 Human Coiled-coil NO: 64 domain-containing protein 93 protein sequence fragment SCTAADTAAQITQR SEQ ID Q29960 Human HLA class I NO: 65 histocompatibility antigen, Cw-16 alpha chain protein sequence fragment SGGGGNFVLSTSLVGYLR SEQ ID Q9UKG9 Human Peroxisomal NO: 66 carnitine o- octanoyltransferase protein sequence fragment SGTSIPSAGK SEQ ID Q7Z5P9 Human Mucin-19 protein NO: 67 sequence fragment SLAGPAGAAPAAPGLGAAAAAPGA SEQ ID Q8NES3 Human Beta-1,3-N- LVR NO: 68 acetylglucosaminyl- transferase lunatic fringe protein sequence fragment SLFPPWTFQFQSGDLEEK SEQ ID Q8NDH2 Human coiled-coil NO: 69 domain-containing protein 168 protein sequence fragment SLGGAVGSVASGAR SEQ ID Q8NHG8 Human E3 ubiquitin- NO:70 protein ligase ZNRF2 protein sequence fragment SPVTFLDSLR SEQ ID A5YKK6 CCR4-NOT transcription NO: 71 complex subunit 1 protein sequence fragment STLVHSLFLTDLYK SEQ ID Q99719 Human Septin-5 protein NO: 72 sequence fragment VEELIESEAPPK SEQ ID Q96B23 Human Uncharacterized NO: 73 protein C18orf25 protein sequence fragment VSELEDFINGPNNAHIQQVGDR SEQ ID P53675 Human Clathrin heavy NO: 74 chain 2 protein sequence fragment YISDLK SEQ ID P55774 Human C-C motif NO: 75 chemokine 18 protein sequence fragment

[0170] The compositions and methods of the appended claims are not limited in scope by the specific compositions and methods described herein, which are intended as illustrations of a few aspects of the claims. Any compositions and methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the compositions and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative compositions and method steps disclosed herein are specifically described, other combinations of the compositions and method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein or less, however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated.

[0171] The term "comprising" and variations thereof as used herein is used synonymously with the term "including" and variations thereof and are open, non-limiting terms. Although the terms "comprising" and "including" have been used herein to describe various embodiments, the terms "consisting essentially of" and "consisting of" can be used in place of "comprising" and "including" to provide for more specific embodiments of the invention and are also disclosed. Other than where noted, all numbers expressing geometries, dimensions, and so forth used in the specification and claims are to be understood at the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, to be construed in light of the number of significant digits and ordinary rounding approaches.

[0172] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

Sequence CWU 1

1

751153PRTHomo sapiensMISC_FEATURE(107)..(107)Beta-N-Methylamino-L-AlanineMISC_FEATURE(1- 07)..(107) 1Ala Thr Lys Ala Val Cys Val Leu Lys Gly Asp Gly Pro Val Gln Gly1 5 10 15Ile Ile Asn Phe Glu Gln Lys Glu Ser Asn Gly Pro Val Lys Val Trp 20 25 30Gly Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe His Val His 35 40 45Glu Phe Gly Asp Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro His Phe 50 55 60Asn Pro Leu Ser Arg Lys His Gly Gly Pro Lys Asp Glu Glu Arg His65 70 75 80Val Gly Asp Leu Gly Asn Val Thr Ala Asp Lys Asp Gly Val Ala Asp 85 90 95Val Ser Ile Glu Asp Ser Val Ile Ser Leu Xaa Gly Asp His Cys Ile 100 105 110Ile Gly Arg Thr Leu Val Val His Glu Lys Ala Asp Asp Leu Gly Lys 115 120 125Gly Gly Asn Glu Glu Ser Thr Lys Thr Gly Asn Ala Gly Ser Arg Leu 130 135 140Ala Cys Gly Val Ile Gly Ile Ala Gln145 150224PRTHomo sapiensMISC_FEATURE(16)..(16)Beta-N-Methylamino-L-AlanineMISC_FEATURE(20)- ..(20)Carbamidomethylated Cysteine 2Asp Gly Val Ala Asp Val Ser Ile Glu Asp Ser Val Ile Ser Leu Xaa1 5 10 15Gly Asp His Cys Ile Ile Gly Arg 20336PRTHomo sapiensMISC_FEATURE(28)..(28)Beta-N-Methylamino-L-AlanineMISC_FEATURE(32)- ..(32)Carbamidomethylated Cysteine 3His Val Gly Asp Leu Gly Asn Val Thr Ala Asp Lys Asp Gly Val Ala1 5 10 15Asp Val Ser Ile Glu Asp Ser Val Ile Ser Leu Xaa Gly Asp His Cys 20 25 30Ile Ile Gly Arg 354153PRTHomo sapiens 4Ala Thr Lys Ala Val Cys Val Leu Lys Gly Asp Gly Pro Val Gln Gly1 5 10 15Ile Ile Asn Phe Glu Gln Lys Glu Ser Asn Gly Pro Val Lys Val Trp 20 25 30Gly Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe His Val His 35 40 45Glu Phe Gly Asp Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro His Phe 50 55 60Asn Pro Leu Ser Arg Lys His Gly Gly Pro Lys Asp Glu Glu Arg His65 70 75 80Val Gly Asp Leu Gly Asn Val Thr Ala Asp Lys Asp Gly Val Ala Asp 85 90 95Val Ser Ile Glu Asp Ser Val Ile Ser Leu Ser Gly Asp His Cys Ile 100 105 110Ile Gly Arg Thr Leu Val Val His Glu Lys Ala Asp Asp Leu Gly Lys 115 120 125Gly Gly Asn Glu Glu Ser Thr Lys Thr Gly Asn Ala Gly Ser Arg Leu 130 135 140Ala Cys Gly Val Ile Gly Ile Ala Gln145 1505154PRTHomo sapiensMISC_FEATURE(108)..(108)Beta-N-Methylamino-L-Alanine 5Met Ala Thr Lys Ala Val Cys Val Leu Lys Gly Asp Gly Pro Val Gln1 5 10 15Gly Ile Ile Asn Phe Glu Gln Lys Glu Ser Asn Gly Pro Val Lys Val 20 25 30Trp Gly Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe His Val 35 40 45His Glu Phe Gly Asp Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro His 50 55 60Phe Asn Pro Leu Ser Arg Lys His Gly Gly Pro Lys Asp Glu Glu Arg65 70 75 80His Val Gly Asp Leu Gly Asn Val Thr Ala Asp Lys Asp Gly Val Ala 85 90 95Asp Val Ser Ile Glu Asp Ser Val Ile Ser Leu Xaa Gly Asp His Cys 100 105 110Ile Ile Gly Arg Thr Leu Val Val His Glu Lys Ala Asp Asp Leu Gly 115 120 125Lys Gly Gly Asn Glu Glu Ser Thr Lys Thr Gly Asn Ala Gly Ser Arg 130 135 140Leu Ala Cys Gly Val Ile Gly Ile Ala Gln145 1506154PRTHomo sapiens 6Met Ala Thr Lys Ala Val Cys Val Leu Lys Gly Asp Gly Pro Val Gln1 5 10 15Gly Ile Ile Asn Phe Glu Gln Lys Glu Ser Asn Gly Pro Val Lys Val 20 25 30Trp Gly Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe His Val 35 40 45His Glu Phe Gly Asp Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro His 50 55 60Phe Asn Pro Leu Ser Arg Lys His Gly Gly Pro Lys Asp Glu Glu Arg65 70 75 80His Val Gly Asp Leu Gly Asn Val Thr Ala Asp Lys Asp Gly Val Ala 85 90 95Asp Val Ser Ile Glu Asp Ser Val Ile Ser Leu Ser Gly Asp His Cys 100 105 110Ile Ile Gly Arg Thr Leu Val Val His Glu Lys Ala Asp Asp Leu Gly 115 120 125Lys Gly Gly Asn Glu Glu Ser Thr Lys Thr Gly Asn Ala Gly Ser Arg 130 135 140Leu Ala Cys Gly Val Ile Gly Ile Ala Gln145 150723PRTHomo sapiens 7Ala Thr Lys Ala Val Cys Val Leu Lys Gly Asp Gly Pro Val Gln Gly1 5 10 15Ile Ile Asn Phe Glu Gln Lys 20820PRTHomo sapiens 8Ala Val Cys Val Leu Lys Gly Asp Gly Pro Val Gln Gly Ile Ile Asn1 5 10 15Phe Glu Gln Lys 20933PRTHomo sapiens 9Ala Val Cys Val Leu Lys Gly Asp Gly Pro Val Gln Gly Ile Ile Asn1 5 10 15Phe Glu Gln Lys Glu Ser Asn Gly Pro Val Lys Val Trp Gly Ser Ile 20 25 30Lys1014PRTHomo sapiens 10Ala Asp Asp Leu Gly Lys Gly Gly Asn Glu Glu Ser Thr Lys1 5 101113PRTHomo sapiens 11Glu Ser Asn Gly Pro Val Lys Val Trp Gly Ser Ile Lys1 5 101233PRTHomo sapiens 12Gly Leu Thr Glu Gly Leu His Gly Phe His Val His Glu Phe Gly Asp1 5 10 15Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro His Phe Asn Pro Leu Ser 20 25 30Arg1312PRTHomo sapiens 13His Val Gly Asp Leu Gly Asn Val Thr Ala Asp Lys1 5 101436PRTHomo sapiens 14His Val Gly Asp Leu Gly Asn Val Thr Ala Asp Lys Asp Gly Val Ala1 5 10 15Asp Val Ser Ile Glu Asp Ser Val Ile Ser Leu Ser Gly Asp His Cys 20 25 30Ile Ile Gly Arg 351540PRTHomo sapiens 15Asp Glu Glu Arg His Val Gly Asp Leu Gly Asn Val Thr Ala Asp Lys1 5 10 15Asp Gly Val Ala Asp Val Ser Ile Glu Asp Ser Val Ile Ser Leu Ser 20 25 30Gly Asp His Cys Ile Ile Gly Arg 35 401631PRTHomo sapiens 16Asp Phe Val Ala Asp Val Ser Ile Glu Asp Ser Val Ile Ser Leu Ser1 5 10 15Gly Asp His Cys Ile Ile Gly Arg Thr Leu Val Val His Glu Lys 20 25 301724PRTHomo sapiens 17Asp Gly Val Ala Asp Val Ser Ile Glu Asp Ser Val Ile Ser Leu Ser1 5 10 15Gly Asp His Cys Ile Ile Gly Arg 201813PRTHomo sapiens 18Thr Leu Val Val His Glu Lys Ala Asp Asp Leu Gly Lys1 5 101928PRTHomo sapiens 19Thr Leu Val Val His Glu Lys Ala Asp Asp Leu Gly Lys Gly Gly Asn1 5 10 15Glu Glu Ser Thr Lys Thr Gly Asn Ala Gly Ser Arg 20 252016PRTHomo sapiens 20Gly Gly Asn Glu Glu Ser Thr Lys Thr Gly Asn Ala Ser Gly Ser Arg1 5 10 152117PRTHomo sapiens 21Thr Gly Asn Ala Gly Ser Arg Leu Ala Cys Gly Val Ile Gly Ile Ala1 5 10 15Gln2239PRTHomo sapiens 22Val Trp Gly Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe His1 5 10 15Val His Glu Phe Gly Asp Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro 20 25 30His Phe Asn Pro Leu Ser Arg 352336PRTHomo sapiens 23His Val Gly Lys Leu Gly Asn Val Thr Ala Asp Lys Asp Gly Val Ala1 5 10 15Asp Val Ser Ile Glu Asp Ser Val Ile Ser Leu Ser Gly Asp His Cys 20 25 30Ile Ile Gly Arg 352424PRTHomo sapiensMISC_FEATURE(14)..(14)Serine conversion to B-N-Methylamino-L-AlanineMISC_FEATURE(15)..(15)Carbamidomethylation 24Ala Tyr His Glu Gln Leu Ser Val Ala Glu Ile Thr Ser Xaa Cys Phe1 5 10 15Glu Pro Asn Ser Gln Met Val Lys 202521PRTHomo sapiensMISC_FEATURE(12)..(12)Serine conversion to B-N-Methylamino-L-Alanine 25Asp Leu Tyr Ala Asn Thr Val Leu Ser Gly Gly Xaa Thr Met Tyr Pro1 5 10 15Gly Ile Ala Asp Arg 20269PRTHomo sapiensMISC_FEATURE(4)..(4)CrotonaldehydeMISC_FEATURE(7)..(7)Serine conversion to B-N-Methylamino-L-Alanine 26Asp Thr Ile Cys Glu Glu Xaa Leu Arg1 5278PRTHomo sapiensMISC_FEATURE(6)..(6)Serine conversion to B-N-Methylamino-L-Alanine 27Glu Ser Val Ser Ser Xaa Asp Arg1 52814PRTHomo sapiensMISC_FEATURE(11)..(11)Serine conversion to B-N-Methylamino-L-Alanine 28Gly Tyr Glu Cys Ile Leu Asn Ile Gln Gly Xaa Glu Gln Arg1 5 102927PRTHomo sapiensMISC_FEATURE(19)..(19)Serine conversion to B-N-Methylamino-L-Alanine 29His Thr Gly Pro Gly Leu Leu Ser Met Ala Asn Ser Gly Pro Asn Thr1 5 10 15Asn Gly Xaa Gln Phe Phe Leu Thr Cys Asp Lys 20 253021PRTHomo sapiensMISC_FEATURE(9)..(9)Serine conversion to B-N-Methylamino-L-Alanine 30Lys Ala Thr Tyr Val Tyr Glu Thr Xaa Gly Pro Asn Leu Ser Asp Asn1 5 10 15Lys Ser Gly Gln Lys 203114PRTHomo sapiensMISC_FEATURE(5)..(5)Serine conversion to B-N-Methylamino-L-Alanine 31Lys Gly Met Trp Xaa Glu Gly Asn Gly Ser His Thr Ile Arg1 5 103211PRTHomo sapiensMISC_FEATURE(3)..(3)Serine conversion to B-N-Methylamino-L-Alanine 32Lys Thr Xaa Thr Asp Phe Ser Glu Val Ile Lys1 5 103310PRTHomo sapiensMISC_FEATURE(5)..(5)Serine conversion to B-N-Methylamino-L-Alanine 33Leu Gly Gln Gly Xaa Gly Gln Gly Pro Lys1 5 103410PRTHomo sapiensMISC_FEATURE(6)..(6)Serine conversion to B-N-Methylamino-L-Alanine 34Leu Leu Leu Gly Thr Xaa Gly Glu Gly Lys1 5 103516PRTHomo sapiensMISC_FEATURE(9)..(9)Serine conversion to B-N-Methylamino-L-Alanine 35Leu Ser Leu Met Leu Asp Glu Gly Xaa Ser Cys Pro Thr Pro Ala Lys1 5 10 153614PRTHomo sapiensMISC_FEATURE(12)..(12)Serine conversion to B-N-Methylamino-L-Alanine 36Met Ser Asp Ile Leu Arg Glu Leu Leu Cys Val Xaa Glu Lys1 5 103711PRTHomo sapiensMISC_FEATURE(4)..(4)Serine conversion to B-N-Methylamino-L-Alanine 37Asn Thr Ala Xaa His Thr Ala Ala Ala Ala Arg1 5 103812PRTHomo sapiensMISC_FEATURE(2)..(2)Serine conversion to B-N-Methylamino-L-Alanine 38Gln Xaa Glu Leu Ser Ala Glu Glu Ser Pro Glu Lys1 5 103914PRTHomo sapiensMISC_FEATURE(1)..(1)Serine conversion to B-N-Methylamino-L-AlanineMISC_FEATURE(2)..(2)Crotonaldehyde 39Xaa Cys Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln Arg1 5 104018PRTHomo sapiensMISC_FEATURE(10)..(10)Serine conversion to B-N-Methylamino-L-Alanine 40Ser Gly Gly Gly Gly Asn Phe Val Leu Xaa Thr Ser Leu Val Gly Tyr1 5 10 15Leu Arg4110PRTHomo sapiensMISC_FEATURE(1)..(1)Serine conversion to B-N-Methylamino-L-Alanine 41Xaa Gly Thr Ser Ile Pro Ser Ala Gly Lys1 5 104226PRTHomo sapiensMISC_FEATURE(1)..(1)Serine conversion to B-N-Methylamino-L-Alanine 42Xaa Leu Ala Gly Pro Ala Gly Ala Ala Pro Ala Pro Gly Leu Gly Ala1 5 10 15Ala Ala Ala Ala Pro Gly Ala Leu Val Arg 20 254318PRTHomo sapiensMISC_FEATURE(12)..(12)Serine conversion to B-N-Methylamino-L-Alanine 43Ser Leu Phe Pro Pro Trp Thr Phe Gln Phe Gln Xaa Gly Asp Leu Glu1 5 10 15Glu Lys4414PRTHomo sapiensMISC_FEATURE(11)..(11)Serine conversion to B-N-Methylamino-L-Alanine 44Ser Leu Gly Gly Ala Val Gly Ser Val Ala Xaa Gly Ala Arg1 5 104510PRTHomo sapiensMISC_FEATURE(1)..(1)Serine conversion to B-N-Methylamino-L-Alanine 45Xaa Pro Val Thr Phe Leu Ser Asp Leu Arg1 5 104614PRTHomo sapiensMISC_FEATURE(1)..(1)Serine conversion to B-N-Methylamino-L-Alanine 46Xaa Thr Leu Val His Ser Leu Phe Leu Thr Asp Leu Tyr Lys1 5 104712PRTHomo sapiensMISC_FEATURE(7)..(7)Serine conversion to B-N-Methylamino-L-Alanine 47Val Glu Glu Leu Ile Glu Xaa Glu Ala Pro Pro Lys1 5 104822PRTHomo sapiensMISC_FEATURE(2)..(2)Serine conversion to B-N-Methylamino-L-Alanine 48Val Xaa Glu Leu Glu Asp Phe Ile Asn Gly Pro Asn Asn Ala His Ile1 5 10 15Gln Gln Val Gly Asp Arg 20496PRTHomo sapiensMISC_FEATURE(3)..(3)Serine conversion to B-N-Methylamino-L-Alanine 49Tyr Ile Xaa Asp Leu Lys1 55024PRTHomo sapiens 50Ala Tyr His Glu Gln Leu Ser Val Ala Glu Ile Thr Ser Ser Cys Phe1 5 10 15Glu Pro Asn Ser Gln Met Val Lys 205121PRTHomo sapiens 51Asp Leu Tyr Ala Asn Thr Val Leu Ser Gly Gly Ser Thr Met Tyr Pro1 5 10 15Gly Ile Ala Asp Arg 20529PRTHomo sapiens 52Asp Thr Ile Cys Glu Glu Ser Leu Arg1 5538PRTHomo sapiens 53Glu Ser Val Ser Ser Ser Asp Arg1 55414PRTHomo sapiens 54Gly Tyr Glu Cys Ile Leu Asn Ile Gln Gly Ser Glu Gln Arg1 5 105527PRTHomo sapiens 55His Thr Gly Pro Gly Leu Leu Ser Met Ala Asn Ser Gly Pro Asn Thr1 5 10 15Asn Gly Ser Gln Phe Phe Leu Thr Cys Asp Lys 20 255621PRTHomo sapiens 56Lys Ala Thr Tyr Val Tyr Glu Thr Ser Gly Pro Asn Leu Ser Asp Asn1 5 10 15Lys Ser Gly Gln Lys 205714PRTHomo sapiens 57Lys Gly Met Trp Ser Glu Gly Asn Gly Ser His Thr Ile Arg1 5 105811PRTHomo sapiens 58Lys Thr Ser Thr Asp Phe Ser Glu Val Ile Lys1 5 105910PRTHomo sapiens 59Leu Gly Gln Gly Ser Gly Gln Gly Pro Lys1 5 106010PRTHomo sapiens 60Leu Leu Leu Gly Thr Ser Gly Glu Gly Lys1 5 106116PRTHomo sapiens 61Leu Ser Leu Met Leu Asp Glu Gly Ser Ser Cys Pro Thr Pro Ala Lys1 5 10 156214PRTHomo sapiens 62Met Ser Asp Ile Leu Arg Glu Leu Leu Cys Val Ser Glu Lys1 5 106311PRTHomo sapiens 63Asn Thr Ala Ser His Thr Ala Ala Ala Ala Arg1 5 106412PRTHomo sapiens 64Gln Ser Glu Leu Ser Ala Glu Glu Ser Pro Glu Lys1 5 106514PRTHomo sapiens 65Ser Cys Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln Arg1 5 106618PRTHomo sapiens 66Ser Gly Gly Gly Gly Asn Phe Val Leu Ser Thr Ser Leu Val Gly Tyr1 5 10 15Leu Arg6710PRTHomo sapiens 67Ser Gly Thr Ser Ile Pro Ser Ala Gly Lys1 5 106827PRTHomo sapiens 68Ser Leu Ala Gly Pro Ala Gly Ala Ala Pro Ala Ala Pro Gly Leu Gly1 5 10 15Ala Ala Ala Ala Ala Pro Gly Ala Leu Val Arg 20 256918PRTHomo sapiens 69Ser Leu Phe Pro Pro Trp Thr Phe Gln Phe Gln Ser Gly Asp Leu Glu1 5 10 15Glu Lys7014PRTHomo sapiens 70Ser Leu Gly Gly Ala Val Gly Ser Val Ala Ser Gly Ala Arg1 5 107110PRTHomo sapiens 71Ser Pro Val Thr Phe Leu Asp Ser Leu Arg1 5 107214PRTHomo sapiens 72Ser Thr Leu Val His Ser Leu Phe Leu Thr Asp Leu Tyr Lys1 5 107312PRTHomo sapiens 73Val Glu Glu Leu Ile Glu Ser Glu Ala Pro Pro Lys1 5 107422PRTHomo sapiens 74Val Ser Glu Leu Glu Asp Phe Ile Asn Gly Pro Asn Asn Ala His Ile1 5 10 15Gln Gln Val Gly Asp Arg 20756PRTHomo sapiens 75Tyr Ile Ser Asp Leu Lys1 5

Claims

1. A polypeptide comprising a sequence that is at least 85% identical to SEQ ID NO: 1, wherein amino acid position 107 comprises a BMAA residue which is isotopically labeled and is defined by the formula below ##STR00036## wherein the .sup.13C isotopic enrichment factor for .sup.dC is at least 25; and wherein the .sup.15N isotopic enrichment factor for .sup.fN is at least 100.

2. The polypeptide of claim 1, wherein the .sup.13C isotopic enrichment factor for .sup.dC is at least 80 and the .sup.15N isotopic enrichment factor for .sup.fN is at least 200.

3. The polypeptide of claim 1, wherein the .sup.13C isotopic enrichment factor for .sup.aC, .sup.bC, and .sup.cC is at least 25.

4. The polypeptide of claim 1, wherein the .sup.13C isotopic enrichment factor for .sup.aC, .sup.bC, and .sup.cC is at least 80.

5. The polypeptide of claim 1, wherein the .sup.15N isotopic enrichment factor for .sup.eN is at least 100.

6. The polypeptide of claim 1, wherein the .sup.15N isotopic enrichment factor for .sup.eN is at least 200.

7. The polypeptide of claim 1, wherein the sequence is at least 90% identical to SEQ ID NO: 1.

8. The polypeptide of claim 1, wherein the sequence is at least 95% identical to SEQ ID NO: 1.

9. The polypeptide of claim 1, wherein the sequence is at least 99% identical to SEQ ID NO: 1.

10. The polypeptide of claim 1, wherein the sequence is identical to SEQ ID NO: 1.

11. A polypeptide comprising a sequence that is at least 85% identical to SEQ ID NO: 2, wherein amino acid position 107 comprises a BMAA residue which is isotopically labeled and is defined by the formula below ##STR00037## wherein the .sup.13C isotopic enrichment factor for .sup.dC is at least 25; and wherein the .sup.15N isotopic enrichment factor for .sup.fN is at least 100.

12. The polypeptide of claim 11, wherein the .sup.13C isotopic enrichment factor for .sup.dC is at least 80 and the .sup.15N isotopic enrichment factor for .sup.fN is at least 200.

13. The polypeptide of claim 11, wherein the .sup.13C isotopic enrichment factor for .sup.aC, .sup.bC, and .sup.cC is at least 25.

14. The polypeptide of claim 11, wherein the .sup.13C isotopic enrichment factor for .sup.aC, .sup.bC, and .sup.cC is at least 80.

15. The polypeptide of claim 11, wherein the .sup.15N isotopic enrichment factor for .sup.eN is at least 100.

16. The polypeptide of claim 11, wherein the .sup.15N isotopic enrichment factor for .sup.eN is at least 200.

17. The polypeptide of claim 11, wherein the sequence is at least 90% identical to SEQ ID NO: 2.

18. The polypeptide of claim 11, wherein the sequence is at least 95% identical to SEQ ID NO: 2.

19. The polypeptide of claim 11, wherein the sequence is at least 99% identical to SEQ ID NO: 2.

20. The polypeptide of claim 11, wherein the sequence is identical to SEQ ID NO: 2.

21.-111. (canceled)