Absolute quantitative analysis of intact and oxidized amino acids by LC-MS without prior derivatization

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Absolute quantitative analysis of intact and oxidized amino acids by LC-MS without prior derivatization. / Gamon, Luke F.; Guo, Chaorui; He, Jianfei; Hägglund, Per; Hawkins, Clare L.; Davies, Michael J.

I: Redox Biology, Bind 36, 101586, 09.2020.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningfagfællebedømt

Harvard

Gamon, LF, Guo, C, He, J, Hägglund, P, Hawkins, CL & Davies, MJ 2020, 'Absolute quantitative analysis of intact and oxidized amino acids by LC-MS without prior derivatization', Redox Biology, bind 36, 101586. https://doi.org/10.1016/j.redox.2020.101586

APA

Gamon, L. F., Guo, C., He, J., Hägglund, P., Hawkins, C. L., & Davies, M. J. (2020). Absolute quantitative analysis of intact and oxidized amino acids by LC-MS without prior derivatization. Redox Biology, 36, [101586]. https://doi.org/10.1016/j.redox.2020.101586

Vancouver

Gamon LF, Guo C, He J, Hägglund P, Hawkins CL, Davies MJ. Absolute quantitative analysis of intact and oxidized amino acids by LC-MS without prior derivatization. Redox Biology. 2020 sep;36. 101586. https://doi.org/10.1016/j.redox.2020.101586

Author

Gamon, Luke F. ; Guo, Chaorui ; He, Jianfei ; Hägglund, Per ; Hawkins, Clare L. ; Davies, Michael J. / Absolute quantitative analysis of intact and oxidized amino acids by LC-MS without prior derivatization. I: Redox Biology. 2020 ; Bind 36.

Bibtex

@article{9f45e4020fcf4971a71f0a97ac40c211,
title = "Absolute quantitative analysis of intact and oxidized amino acids by LC-MS without prior derivatization",
abstract = "The precise characterization and quantification of oxidative protein damage is a significant challenge due to the low abundance, large variety, and heterogeneity of modifications. Mass spectrometry (MS)-based techniques at the peptide level (proteomics) provide a detailed but limited picture due to incomplete sequence coverage and imperfect enzymatic digestion. This is particularly problematic with oxidatively modified and cross-linked/aggregated proteins. There is a pressing need for methods that can quantify large numbers of modified amino acids, which are often present in low abundance compared to the high background of non-damaged amino acids, in a rapid and reliable fashion. We have developed a protocol using zwitterionic ion-exchange chromatography coupled with LC-MS to simultaneously quantify both parent amino acids and their respective oxidation products. Proteins are hydrolyzed with methanesulfonic acid in the presence of tryptamine and purified by strong cation exchange solid phase extraction. The method was validated for the common amino acids (excluding Gln, Asn, Cys) and the oxidation products 3-chlorotyrosine (3-ClTyr), 3-nitrotyrosine (3-NO2Tyr), di-tyrosine, Nε-(1-carboxymethyl)-L-lysine, o,o’-di-tyrosine, 3,4,-dihydroxyphenylalanine, hydroxy-tryptophan and kynurenine. Linear standard curves were observed over ~3 orders of magnitude dynamic range (2–1000 pmol for parent amino acids, 80 fmol–20 pmol for oxidation products) with limit-of-quantification values as low as 200 fmol (o,o’-di-tyrosine). The validated method was used to quantify Tyr and Trp loss, and formation of 3-NO2Tyr on the isolated protein anastellin treated with peroxynitrous acid, and for 3-ClTyr formation (over a 2 orders of magnitude range) in cell lysates and complex protein mixtures treated with hypochlorous acid.",
keywords = "3-Chlorotyrosine, 3-Nitrotyrosine, Chlorination, Hypochlorous acid, LC-MS, Methionine sulfoxide, Nitration, Peroxynitrite, Post-translational modifications, Protein oxidation",
author = "Gamon, {Luke F.} and Chaorui Guo and Jianfei He and Per H{\"a}gglund and Hawkins, {Clare L.} and Davies, {Michael J.}",
year = "2020",
month = "9",
doi = "10.1016/j.redox.2020.101586",
language = "English",
volume = "36",
journal = "Redox Biology",
issn = "2213-2317",
publisher = "Elsevier",

}

RIS

TY - JOUR

T1 - Absolute quantitative analysis of intact and oxidized amino acids by LC-MS without prior derivatization

AU - Gamon, Luke F.

AU - Guo, Chaorui

AU - He, Jianfei

AU - Hägglund, Per

AU - Hawkins, Clare L.

AU - Davies, Michael J.

PY - 2020/9

Y1 - 2020/9

N2 - The precise characterization and quantification of oxidative protein damage is a significant challenge due to the low abundance, large variety, and heterogeneity of modifications. Mass spectrometry (MS)-based techniques at the peptide level (proteomics) provide a detailed but limited picture due to incomplete sequence coverage and imperfect enzymatic digestion. This is particularly problematic with oxidatively modified and cross-linked/aggregated proteins. There is a pressing need for methods that can quantify large numbers of modified amino acids, which are often present in low abundance compared to the high background of non-damaged amino acids, in a rapid and reliable fashion. We have developed a protocol using zwitterionic ion-exchange chromatography coupled with LC-MS to simultaneously quantify both parent amino acids and their respective oxidation products. Proteins are hydrolyzed with methanesulfonic acid in the presence of tryptamine and purified by strong cation exchange solid phase extraction. The method was validated for the common amino acids (excluding Gln, Asn, Cys) and the oxidation products 3-chlorotyrosine (3-ClTyr), 3-nitrotyrosine (3-NO2Tyr), di-tyrosine, Nε-(1-carboxymethyl)-L-lysine, o,o’-di-tyrosine, 3,4,-dihydroxyphenylalanine, hydroxy-tryptophan and kynurenine. Linear standard curves were observed over ~3 orders of magnitude dynamic range (2–1000 pmol for parent amino acids, 80 fmol–20 pmol for oxidation products) with limit-of-quantification values as low as 200 fmol (o,o’-di-tyrosine). The validated method was used to quantify Tyr and Trp loss, and formation of 3-NO2Tyr on the isolated protein anastellin treated with peroxynitrous acid, and for 3-ClTyr formation (over a 2 orders of magnitude range) in cell lysates and complex protein mixtures treated with hypochlorous acid.

AB - The precise characterization and quantification of oxidative protein damage is a significant challenge due to the low abundance, large variety, and heterogeneity of modifications. Mass spectrometry (MS)-based techniques at the peptide level (proteomics) provide a detailed but limited picture due to incomplete sequence coverage and imperfect enzymatic digestion. This is particularly problematic with oxidatively modified and cross-linked/aggregated proteins. There is a pressing need for methods that can quantify large numbers of modified amino acids, which are often present in low abundance compared to the high background of non-damaged amino acids, in a rapid and reliable fashion. We have developed a protocol using zwitterionic ion-exchange chromatography coupled with LC-MS to simultaneously quantify both parent amino acids and their respective oxidation products. Proteins are hydrolyzed with methanesulfonic acid in the presence of tryptamine and purified by strong cation exchange solid phase extraction. The method was validated for the common amino acids (excluding Gln, Asn, Cys) and the oxidation products 3-chlorotyrosine (3-ClTyr), 3-nitrotyrosine (3-NO2Tyr), di-tyrosine, Nε-(1-carboxymethyl)-L-lysine, o,o’-di-tyrosine, 3,4,-dihydroxyphenylalanine, hydroxy-tryptophan and kynurenine. Linear standard curves were observed over ~3 orders of magnitude dynamic range (2–1000 pmol for parent amino acids, 80 fmol–20 pmol for oxidation products) with limit-of-quantification values as low as 200 fmol (o,o’-di-tyrosine). The validated method was used to quantify Tyr and Trp loss, and formation of 3-NO2Tyr on the isolated protein anastellin treated with peroxynitrous acid, and for 3-ClTyr formation (over a 2 orders of magnitude range) in cell lysates and complex protein mixtures treated with hypochlorous acid.

KW - 3-Chlorotyrosine

KW - 3-Nitrotyrosine

KW - Chlorination

KW - Hypochlorous acid

KW - LC-MS

KW - Methionine sulfoxide

KW - Nitration

KW - Peroxynitrite

KW - Post-translational modifications

KW - Protein oxidation

UR - http://www.scopus.com/inward/record.url?scp=85085708451&partnerID=8YFLogxK

U2 - 10.1016/j.redox.2020.101586

DO - 10.1016/j.redox.2020.101586

M3 - Journal article

C2 - 32505089

AN - SCOPUS:85085708451

VL - 36

JO - Redox Biology

JF - Redox Biology

SN - 2213-2317

M1 - 101586

ER -

ID: 243999625