A Robust Method for Quantitative High-throughput Analysis of Proteomes by 18O Labeling

Abstract

MS-based quantitative proteomics plays an increasingly important role in biological and medical research and the development of these techniques remains one of the most important challenges in mass spectrometry. Numerous stable isotope labeling approaches have been proposed. However, and particularly in the case of 18 O-labeling, a standard protocol of general applicability is still lacking, and statistical issues associated to these methods remain to be investigated. In this work we present an improved high-throughput quantitative proteomics method based on whole proteome concentration by SDS-PAGE, optimized in-gel digestion, peptide 18 O-labeling, and separation by off-gel isoelectric focusing followed by liquid chromatography-LIT-MS. We demonstrate that the off-gel technique is fully compatible with 18 The analysis of differential protein expression is fundamental for the understanding of biological processes and plays an increasingly important role in biological and medical research (1). In recent years, numerous stable isotope labeling (SIL) 1 techniques have emerged as alternatives to the historically used two-dimensional-based approaches for semiquantitative proteomic studies. In these techniques the quantification is done in the same mass spectrometer where peptides are analyzed by tandem mass spectrometry (MS/MS), so relative quantification and peptide identification is performed at the same time. The differences among the several existing SIL approaches are mainly related to the way labels are introduced and the method used to perform the quantification by MS. Thus, in the SILAC method (2) labels are introduced metabolically at the protein level before peptides are generated from protein by enzymatic digestion, minimizing variability introduced by peptide preparation, whereas in the others labeling is performed postdigestion at the peptide level, either chemically in the iTRAQ method (3), or enzymatically in the 18 O labeling method (4 -6). In the iTRAQ method, quantification is made at the MS/MS level, allowing the possibility of performing multiplexed comparisons (7) whereas in SILAC and 18 O methods peptides are quantified at the MS level and are mainly used for pairwise comparisons. In other SIL approaches, such as the ICAT method (8), labeled peptides are specifically recovered after an affinity purification approach; this allows reducing peptide complexity, which is particularly appropriate to selectively analyze peptide subpopulations, such as reduced or oxidized cys-containing peptides (9). The 18 O labeling method has the advantage that labels are intro-

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