title: Comparison of SILAC and mTRAQ quantification for phosphoproteomics on a quadrupole orbitrap mass spectrometer.
abstract: Advances in mass spectrometric methodology and instrumentation have promoted a continuous increase in analytical performance in the field of phosphoproteomics. Here, we employed the recently introduced quadrupole Orbitrap (Q Exactive) mass spectrometer for quantitative signaling analysis to a depth of more than 15 000 phosphorylation sites. In parallel to the commonly used SILAC approach, we evaluated the nonisobaric chemical labeling reagent mTRAQ as an alternative quantification technique. Both enabled high phosphoproteome coverage in H3122 lung cancer cells. Replicate quantifications by mTRAQ identified almost as many significant phosphorylation changes upon treatment with ALK kinase inhibitor crizotinib as found by SILAC quantification. Overall, mTRAQ was slightly less precise than SILAC as evident from a somewhat higher variance of replicate phosphosite ratios. Direct comparison of SILAC- and mTRAQ-quantified phosphosites revealed that the majority of changes were detected by either quantification techniques, but also highlighted the aspect of false negative identifications in quantitative proteomics applications. Further inspection of crizotinib-regulated phosphorylation changes unveiled interference with multiple antioncogenic mechanisms downstream of ALK fusion kinase in H3122 cells. In conclusion, our results demonstrate a strong analytical performance of the Q Exactive in global phosphoproteomics, and establish mTRAQ quantification as a useful alternative to metabolic isotope labeling.
authors.affiliation: Evotec München GmbH, Am Klopferspitz 19a, 82152 Martinsried, Germany.
authors.forename: Felix S
authors.initials: FS
authors.lastname: Oppermann
authors.forename: Martin
authors.initials: M
authors.lastname: Klammer
authors.forename: Caroline
authors.initials: C
authors.lastname: Bobe
authors.forename: Jürgen
authors.initials: J
authors.lastname: Cox
authors.forename: Christoph
authors.initials: C
authors.lastname: Schaab
authors.forename: Andreas
authors.initials: A
authors.lastname: Tebbe
authors.forename: Henrik
authors.initials: H
authors.lastname: Daub
mesh_terms: D000077547:Crizotinib; D006801:Humans; D007553:Isotope Labeling; D020014:K562 Cells; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D060066:Protein Interaction Maps; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D020543:Proteome / Q000737:chemistry* / Q000378:metabolism; D040901:Proteomics; D011720:Pyrazoles / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D053719:Tandem Mass Spectrometry
chemical_list: D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D020543:Proteome; D011720:Pyrazoles; D011725:Pyridines; D000077547:Crizotinib; D011494:Protein Kinases