Estimation of Absolute Protein Quantities of Unlabeled Samples by Selected Reaction Monitoring Mass Spectrometry

Abstract
For many research questions in modern molecular and systems biology information about absolute protein quantities is imperative. These include, for example, kinetic modeling of processes, protein turnover determinations, stoichiometric investigations of protein complexes or quantitative comparisons of different proteins within one or across samples. To date, the vast majority of proteomic studies are limited to providing relative quantitative comparisons of protein levels between limited numbers of samples. Here we describe and demonstrate the utility of a targeting mass spectrometric (MS) technique for the estimation of absolute protein abundance in unlabeled and non-fractionated cell lysates. The method is based on selected reaction monitoring (SRM) mass spectrometry and the best-flyer hypothesis, which assumes that the specific MS signal intensity of the most intense tryptic peptides per protein is approximately constant throughout a whole proteome. SRM targeted best-flyer peptides were selected for each protein from the peptide precursor ion signal intensities from directed MS data. The most-intense transitions per peptide were selected from full MS/MS scans of crude synthetic analogs. We used Monte Carlo cross-validation to systematically investigate the accuracy of the technique as a function of the number of measured best-flyer peptides and the number of SRM transitions per peptide. We find, that a linear model based on the two most-intense transitions of the three best-flying peptides per proteins (TopPep3/TopTra2) generates optimal results with a cross-correlated mean-fold error of 1.8 and a squared Pearson coefficient R2 of 0.88. Applying the optimized model to lysates of the microbe Leptospira interrogans we detected significant protein abundance changes of 39 target proteins upon antibiotic treatment, which correlate well with literature values. The described method is generally applicable and exploits the inherent performance advantages of SRM, such as high sensitivity, selectivity, reproducibility and dynamic range, and estimates absolute protein concentrations of selected proteins at minimized costs.

The article is published online in the journal Molecular & Cellular Proteomics and is free to access.