title: Targeted mass spectrometry methods for detecting oxidative post-translational modifications.
abstract: Oxidative post-translational modifications (oxPTMs) can alter the function of proteins, and are important in the redox regulation of cell behaviour. The most informative technique to detect and locate oxPTMs within proteins is mass spectrometry (MS). However, proteomic MS data are usually searched against theoretical databases using statistical search engines, and the occurrence of unspecified or multiple modifications, or other unexpected features, can lead to failure to detect the modifications and erroneous identifications of oxPTMs. We have developed a new approach for mining data from accurate mass instruments that allows multiple modifications to be examined. Accurate mass extracted ion chromatograms (XIC) for specific reporter ions from peptides containing oxPTMs were generated from standard LC-MSMS data acquired on a rapid-scanning high-resolution mass spectrometer (ABSciex 5600 Triple TOF). The method was tested using proteins from human plasma or isolated LDL. A variety of modifications including chlorotyrosine, nitrotyrosine, kynurenine, oxidation of lysine, and oxidized phospholipid adducts were detected. For example, the use of a reporter ion at 184.074Da/e, corresponding to phosphocholine, was used to identify for the first time intact oxidized phosphatidylcholine adducts on LDL. In all cases the modifications were confirmed by manual sequencing. ApoB-100 containing oxidized lipid adducts was detected even in healthy human samples, as well as LDL from patients with chronic kidney disease. The accurate mass XIC method gave a lower false positive rate than normal database searching using statistical search engines, and identified more oxidatively modified peptides. A major advantage was that additional modifications could be searched after data collection, and multiple modifications on a single peptide identified. The oxPTMs present on albumin and ApoB-100 have potential as indicators of oxidative damage in ageing or inflammatory diseases.
authors.affiliation: Aston University (Birmingham), Life and Health Sciences, United Kingdom.. Electronic address: k.tveen-jensen@aston.ac.uk.
authors.forename: Karina
authors.initials: K
authors.lastname: Tveen-Jensen
authors.affiliation: Aston University (Birmingham), Life and Health Sciences, United Kingdom.
authors.forename: Ana
authors.initials: A
authors.lastname: Reis
authors.affiliation: Aston University (Birmingham), Life and Health Sciences, United Kingdom.
authors.forename: Corinne M
authors.initials: CM
authors.lastname: Spickett
authors.affiliation: Aston University (Birmingham), Life and Health Sciences, United Kingdom.
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Pitt