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

BACKGROUND

The non-protein amino acid β-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 β-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.

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.

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

Provided herein are isotopically labeled reagents, including isotopically labeled small molecules and peptides, that can be used to detect and/or quantify β-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.

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

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wherein the ¹³C isotopic enrichment factor for ^dC is at least 25; and wherein the ¹⁵N isotopic enrichment factor for N is at least 100.

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

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wherein the ¹³C isotopic enrichment factor for ^dC is at least 25; and wherein the ¹⁵N isotopic enrichment factor for N is at least 100.

In another aspect, provided herein is a method for detecting β-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

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In one aspect, provided herein is a method for detecting β-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 β-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.

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

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wherein R represents hydrogen or an amine protecting group, and at least two of ^aC, ^bC, ^cC, ^dC, ^eN, ^fN, ^gO, and ^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.

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 β-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.

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wherein R represents hydrogen or an amine protecting group, and at least two of ^aC, ^bC, ^cC, ^dC, ^eN, ^fN, ^gO, and ^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.

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 β-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

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).

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

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

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

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.

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

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.

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

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

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

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.

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.

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.

FIG. 14 illustrates the tandem mass spectrum of BMAA containing stable isotope-label (¹³C₄¹⁵N₂β-N-Methylamino-L-Alanine, or Compound 3).

DETAILED DESCRIPTION

Provided herein are isotopically labeled reagents, including isotopically labeled small molecules and peptides, that can be used to detect and/or quantify β-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.

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). β-N-methylamino-L-alanine (BMAA) is represented using the letter B in a peptide sequence, unless otherwise indicated in the text.

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

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

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wherein the ¹³C isotopic enrichment factor for ^dC is at least 25; and wherein the ¹⁵N isotopic enrichment factor for N is at least 100.

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

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.

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wherein the ¹³C isotopic enrichment factor for ^dC is at least 25; and wherein the ¹⁵N isotopic enrichment factor for N is at least 100.

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

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.

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.

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.

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

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.

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.

SEQ ID NO: 6 = Human SOD1 wild-type protein

sequence (with initial methionine)

MATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHE

FGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSI

EDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVI

GIAQ

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.

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

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

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

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

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.

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.

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^thEdition, 2001, Wiley-Interscience Publication, New York).

Isotopically labeled amino acids, such as ¹³C/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., ¹³C/¹⁵N-labeled asparagine).

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.

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.

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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 β-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

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

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

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

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

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

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.

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

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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 ¹³C isotopic enrichment factor for ^dC is at least 25; the ¹⁵N isotopic enrichment factor for N is at least 100; and independently for each occurrence in the polypeptide, R₁is H and R₂is selected from one of the following

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or R₁and R₂, together with the atoms to which they are attached, form a five-membered heterocycle defined by the structure below

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

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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 ¹³C isotopic enrichment factor for ^dC is at least 25; the ¹⁵N isotopic enrichment factor for N is at least 100; and independently for each occurrence in the polypeptide, R₁is H and R₂is selected from one of the following

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or R₁and R₂, together with the atoms to which they are attached, form a five-membered heterocycle defined by the structure below

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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).

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).

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).

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.

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.

Also provided are compositions comprising an isotopically labeled polypeptide that includes one or more β-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 (¹³C, ¹⁵N, and/or 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.

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).

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 (¹³C, ¹⁵N, and/or ¹⁸O).

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 β-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 α,β-unsaturated amino acid residue. Once activated, the α,β-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 ¹³C/¹⁵N-labeled methylamine) and Ba(OH)₂(e.g., 0.1 M Ba(OH)₂) in water/DMSO/EtOH (2:2:1) at basic pH (pH 12.5) and elevated temperature (e.g., 37° C.). Once complete, the reaction can be quenched with acid (e.g., acetic acid).

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.

Further provided herein are isotopically labeled reagents, including isotopically labeled small molecules and peptides, that can be used to detect and/or quantify β-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.

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

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

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

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

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

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wherein the ¹³C isotopic enrichment factor for ^dC is at least 25; and wherein the ¹⁵N isotopic enrichment factor for N is at least 100.

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

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wherein the ¹³C isotopic enrichment factor for ^dC is at least 25; and wherein the ¹⁵N isotopic enrichment factor for N is at least 100.

Methods

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.

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.

In one aspect, provided herein is a method for detecting β-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

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

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

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

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

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

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

In one embodiment, the ¹³C isotopic enrichment factor for ^dC is at least 80 and the ¹⁵N isotopic enrichment factor for ^fN is at least 200. In one embodiment, the ¹³C isotopic enrichment factor for ^aC, ^bC, and ^cC is at least 25. In one embodiment, the ¹³C isotopic enrichment factor for ^aC, ^bC, and ^cC is at least 80. In one embodiment, the ¹⁵N isotopic enrichment factor for ^eN is at least 100. In one embodiment, the ¹⁵N isotopic enrichment factor for ^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. In one embodiment, the purified SOD1 protein sample is digested with trypsin.

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

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.

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.

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 β-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.

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

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wherein R represents hydrogen or an amine protecting group, and at least two of ^aC, ^bC, ^cC, ^dC, ^eN, ^fN, ^gO, and ^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.

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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 β-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.

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

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.

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.

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.

In one embodiment, the ALS is sporadic ALS. In one embodiment, the ALS is familial ALS.

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.

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.

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.

The methods herein can also be used to detect β-N-methylamino-L-alanine (BMAA) in additional proteins in a patient sample. In one aspect, provided herein is a method for detecting β-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

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wherein R represents hydrogen or an amine protecting group, and at least two of ^aC, ^bC, ^cC, ^dC, ^eN, ^gO, and ^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.

In one aspect, provided herein is a method for detecting β-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 β-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.

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

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wherein R represents hydrogen or an amine protecting group, and at least two of ^aC, ^bC, ^cC, ^dC, ^eN, ^fN, ^gO, and ^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.

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

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wherein R represents hydrogen or an amine protecting group, and at least two of ^aC, ^bC, ^cC, ^dC, ^eN, ^fN, ^gO, and ^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.

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 β-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.

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.

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

EXAMPLES
Example 1
Preparation of ¹³C₄¹⁵N₂β-N-Methylamino-L-Alanine

Preparation of Compound 1. To a solution of ¹³C-¹⁵N labeled L-asparagine monohydrate (95.8 mg, 0.614 mmol) in 10% aqueous Na₂CO₃(1.6 mL) was added 1,4-dioxane (0.9 mL) and the mixture was cooled to 0° 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 (×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: ¹H NMR (400 MHz, CDCl₃) δ 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]⁺; HRMS m/z calculated for ¹³C₄C₈H₁₄¹⁵N₂O₅[M+Na]⁺ 295.0870, found 295. 0867.

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Preparation of Compound 2. To a slurry of N²-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° C. and the mixture was stirred for 30 min at 15° C. The reaction mixture was then allowed to warm to rt and stirred until completion (4 h). The mixture was cooled to 5° 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]⁺. ¹H NMR (400 MHz, CDCl₃) δ 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]⁺; HRMS m/z calculated for ¹³C₃C₈H₁₄¹⁵N₂O₄[M+H]⁺ 244.1068, found 244.1066.

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Preparation of Compound 3. To a suspension of compound 3 (33.9 mg, 0.139 mmol) in methanol (0.60 mL) was added Et₃N (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° C., followed by the addition of NaBH₄(16.0 mg, 0.416 mmol). The mixture was then stirred for an additional 15 min at 0° C., and concentrated under reduced pressure. The residue was then dissolved in 0.1 M aqueous solution of NaOH, and extracted with diethyl ether (×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 μL, 0.244 mmol) was added. The reaction was stirred for an additional 15 min, and cooled to 0° C. NaBH₄(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 CHCl₃, dried (MgSO₄), 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: ¹H NMR (400 MHz, CDCl₃) δ 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]⁺; HRMS m/z calculated for ¹³C₃C₁₆H₂₂¹⁵N₂O₄[M+H]⁺ 348.1694, found 348.1690.

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Preparation of Compound ¹³C₄¹⁵N₂β-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 H₂gas and stirred under a H₂atmosphere overnight. The Pd/C was filtered through Celite®, 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]⁺; HRMS m/z calculated for ¹³C₃CH₁₀¹⁵N₂O₂[M+H]⁺ 124.0856, found 124.0856. This final product was further characterized by MS/MS to confirm the exact structure (FIG. 14).

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¹³C₄¹⁵N₂β-N-methylamino-L-alanine

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 μL. 20 μg of protein was hydrolyzed using 50 of 6N HCl and incubating at 110° C. for 18 hours. 5 μL of 60.9 mM SIL BMAA was spiked into the samples post-hydrolysis. Samples were then dried and resuspended in 100 μL 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.

The peptide sequence DGVADVSIEDSVISLSGDHCIIGR (SEQ ID NO:17) with highlighted (bold and underlined) Serine phosphorylated, carbamidomethylated Cysteine and ¹³C₆¹⁵N₄isotopically 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 μg) of peptide was added to 20 μL of derivatization solution and incubated at 37° C. for two hours. The reaction was quenched with 2 μL 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.

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° 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° 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×g and 20° 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×g and 20° 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° 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×g and 20° C. 400 uL of quench buffer was added to the retained volume and centrifuged again for 15 minutes at 14,000×g and 20° C. Samples were frozen at −80° 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.

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×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° 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.

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

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).

For the liquid chromatography experiments, the Thermo Easy nanoLC setup included: 75 μm 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 μL of digested (trypsin) SOD1 containing ˜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.

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

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.

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:

1) Dynamic Modifications: Ser→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)
2) SNP Dynamic Modifications: Val→Met, Ala→Val, His→Arg, Thr→Arg, Gly→Arg, Ser→BMAA (Also searched all other amino acids to BMAA)

Fixed: Carbamidomethylation

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

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 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

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

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.

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 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→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→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.

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

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.

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

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→Arg). The additional SNPs identified in Table 5 are novel.

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.

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

Brain tissue samples of 12 matched ALS/healthy control samples were obtained from Emory University. 100× Halt protease/phosphatase inhibitor was diluted to 1× 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 μL of lysis buffer and 750 μg 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 μg 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° 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° C. for 60 minutes. To concentrate the samples, they were spun for 15 minutes at 14,000×g and 20° C. The retained volume was diluted three times with 400 μL of a digesting buffer of 100 mM ammonium bicarbonate at pH 7.0 and spun for 15 minutes at 14,000×g and 20° C. between each buffer addition. Filters were then transferred to a clean tube. 2 μg SIL SOD1 surrogate peptide containing BMAA was reconstituted in 500 μL of 0.001% Zwittergent 3-16, then 5 μL was spiked into each sample. The samples were digested using 45 μL of an enzyme solution containing 100 μg/ml modified trypsin and 100 mM ammonium bicarbonate to give a 1:50 enzyme:protein ratio and incubated overnight at 37° C. 50 μL 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×g to elute the peptides. 400 μL of the quench buffer was added to the retained volume and spun for 15 minutes at 14,000×g using the same collection tube. The samples were lyophilized and kept at −80° C. until analysis. Samples were reconstituted in 100 μL of 0.001% Zwittergent 3-16. 4 μL (approximately 0.5 μg) 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×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° 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:

Serine→BMAA, Carbamidomethylation (Cys), Crotonaldehyde (C,H), and Aspartic acid→Glutamine

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→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 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→BMAA

*C* = Carbamidomethylation or Crotonaldehyde

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

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.

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.

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.

	Number	Date	Country
	62368437	Jul 2016	US
	62368562	Jul 2016	US

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

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Publication Number

Date Filed

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