Benchmarking common quantification strategies for large-scale phosphoproteomics
2018
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 labelingby
amino acidsin
cell culture(SILAC) and MS2- and MS3-measured
tandem mass tags(TMT). In a mixed species comparison with fixed
phosphopeptideratios, 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
phosphoproteomechanges 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.
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