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 -