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

• Published in: Redox biology 2020-09, Vol.36, p.101586-101586, Article 101586
• Main Authors: Gamon, Luke F., Guo, Chaorui, He, Jianfei, Hägglund, Per, Hawkins, Clare L., Davies, Michael J.
• Format: Article
• Language: English
• Subjects: 3-Chlorotyrosine; 3-Nitrotyrosine; Chlorination; Hypochlorous acid; LC-MS; Methionine sulfoxide; Method; Nitration; Peroxynitrite; Post-translational modifications; Protein oxidation
• ISSN: 2213-2317; 2213-2317
• DOI: 10.1016/j.redox.2020.101586

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. [Display omitted] •Identification and quantification of oxidative protein damage is a major challenge.•A versatile LC-MS assay is reported that involves hydrolysis to free amino acids.•Quantification is possible for both parent amino acids and products in single runs.•A dynamic range of 2-3 orders of magnitude is available for most analytes.•Example of use with pure proteins, extracellular matrix and cell lysates are given.