Reporter ion-based mass spectrometry approaches for the detection of non-enzymatic protein modifications in biological samples

Development of mass spectrometry techniques to detect protein oxidation, which contributes to signalling and inflammation, is important. Label-free approaches have the advantage of reduced sample manipulation, but are challenging in complex samples owing to undirected analysis of large data sets using statistical search engines. To identify oxidised proteins in biological samples, we previously developed a targeted approach involving precursor ion scanning for diagnostic MS3 ions from oxidised residues. Here, we tested this approach for other oxidations, and compared it with an alternative approach involving the use of extracted ion chromatograms (XICs) generated from high-resolution MSMS data using very narrow mass windows. This accurate mass XIC data methodology was effective at identifying nitrotyrosine, chlorotyrosine, and oxidative deamination of lysine, and for tyrosine oxidations highlighted more modified peptide species than precursor ion scanning or statistical database searches. Although some false positive peaks still occurred in the XICs, these could be identified by comparative assessment of the peak intensities. The method has the advantage that a number of different modifications can be analysed simultaneously in a single LC-MSMS run. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine. Biological significance: The use of accurate mass extracted product ion chromatograms to detect oxidised peptides could improve the identification of oxidatively damaged proteins in inflammatory conditions. © 2013 Elsevier B.V.

PPD v1.0 - an integrated, web-accessible database of experimentally determined protein pKa values

The Protein pKa Database (PPD) v1.0 provides a compendium of protein residue-specific ionization equilibria (pKa values), as collated from the primary literature, in the form of a web-accessible postgreSQL relational database. Ionizable residues play key roles in the molecular mechanisms that underlie many biological phenomena, including protein folding and enzyme catalysis. The PPD serves as a general protein pKa archive and as a source of data that allows for the development and improvement of pKa prediction systems. The database is accessed through an HTML interface, which offers two fast, efficient search methods: an amino acid-based query and a Basic Local Alignment Search Tool search. Entries also give details of experimental techniques and links to other key databases, such as National Center for Biotechnology Information and the Protein Data Bank, providing the user with considerable background information.