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)O peptide labeling in any pH range. A recently developed statistical model indicated that partial digestions and methionine oxidation do not alter protein quantification and that variances at the scan, peptide, and protein levels are stable and reproducible in a variety of proteomes of different origin. We have also analyzed the dynamic range of quantification and demonstrated the practical utility of the method by detecting expression changes in a model of activation of Jurkat T-cells. Our protocol provides a general approach to perform quantitative proteomics by (18)O-labeling in high-throughput studies, with the added value that it has a validated statistical model for the null hypothesis. To the best of our knowledge, this is the first report where a general protocol for stable isotope labeling is tested in practice using a collection of samples and analyzed at this degree of statistical detail.
Highlights
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 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 18O labeling method (4 – 6)
Quantitative Analysis of Proteomes by One-Dimensional SDS-PAGE Fractionation, In-Gel Digestion and 18O Labeling—We first analyzed whether one-dimensional-SDS-PAGE protein separation was suitable to perform a relative quantification of two protein preparations by 18O labeling
Summary
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]. For the experiments aimed to analyze the effect of activation on cytoplasmic proteins of Jurkat T cells, peptide samples differentially labeled with 16O/18O were desalted separately on C18 Oasis cartridges, mixed at different ratios (1:1, 2:1, 4:1, 8:1, 1:2, 1:4, and 1:8) at a final content of 200 g of peptides and dried-down.
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