Comparison of SILAC and mTRAQ Quantification for Phosphoproteomics on a Quadrupole Orbitrap Mass Spectrometer

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.