Benchmarking common quantification strategies for large-scale phosphoproteomics

Abstract

Comprehensive mass spectrometry (MS)-based proteomics is now feasible, but reproducible quantification remains challenging, especially for post-translational modifications such as phosphorylation. Here, we compare the most popular quantification techniques for global phosphoproteomics: label-free quantification (LFQ), stable isotope labeling by amino acids in cell culture (SILAC) and MS2- and MS3-measured tandem mass tags (TMT). In a mixed species comparison with fixed phosphopeptide ratios, we find LFQ and SILAC to be the most accurate techniques. MS2-based TMT yields the highest precision but lowest accuracy due to ratio compression, which MS3-based TMT can partly rescue. However, MS2-based TMT outperforms MS3-based TMT when analyzing phosphoproteome changes in the DNA damage response, since its higher precision and larger identification numbers allow detection of a greater number of significantly regulated phosphopeptides. Finally, we utilize the TMT multiplexing capabilities to develop an algorithm for determining phosphorylation site stoichiometry, showing that such applications benefit from the high accuracy of MS3-based TMT.

Reproducible workflow for multiplexed deep-scale proteome and phosphoproteome analysis of tumor tissues by liquid chromatography–mass spectrometry

“…For the analyzed datasets, we find the best tradeoff between the highest quantitative reproducibility and the best coverage at two ratio counts for proteins and one ratio count for phosphosites. Isobaric tagging methods have been shown to improve the precision of quantification, whereas label-free methods suffer from high technical variation for PTM applications, in which the majority of quantification events rely on the quantification of single peptides 43 .…”

Full-featured, real-time database searching platform enables fast and accurate multiplexed quantitative proteomics

“…In standard SPS-MS3 methods, these non-matching spectra generate wasteful MS3 scans. We reasoned that elimination of these extraneous SPS-MS3 scans would enhance SPS-MS3 acquisition speed while maintaining quantitative accuracy 3,20 . Therefore, performing RTS prior to SPS-MS3 acquisition and only triggering SPS-MS3 scans when a PSM was observed could significantly reduce the number of spurious SPS-MS3 scans generated 4 .…”

Kinome Profiling of Primary Endometrial Tumors Using Multiplexed Inhibitor Beads and Mass Spectrometry Identifies SRPK1 As Candidate Therapeutic Target

“…Several groups have reported ratio compression arising from contamination during precursor ion selection in MS2 quantitation using isobaric tagging methods, leading to compromised accuracy, compression and underestimation of the ratios (47,48). A potential drawback to label-free quantitation is that experimental conditions such as variations in kinase enrichment from MIB-columns amongst samples, LC/MS run conditions such as temperature, and column conditions may present differences between samples (49). Using labelling techniques such as SILAC or super-SILAC has the benefit of measuring samples within the same MS run (20,24).…”