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.

Large-Scale Label-Free Comparative Proteomics Analysis of Polo-Like Kinase 1 Inhibition via the Small-Molecule Inhibitor BI 6727 (Volasertib) in BRAFV600E Mutant Melanoma Cells

“…The Q Exactive has been shown to identify a higher number of compounds with better confidence in multiple comparison studies. 28−30 Thus, there is greater proteome coverage, which enhances the ability to quantitate lower abundant proteins than is achieved with spectral-counting approaches. 31−36 Indeed, the power of this combination of instrumentation and software was born out in the ability of our analysis to positively identify over 1800 proteins, at a cutoff of ≤1% FDR, with 343 of these identified proteins showing significantly altered expression (Tables S1 and S2 in the Supporting Information).…”

Comparing SILAC- and Stable Isotope Dimethyl-Labeling Approaches for Quantitative Proteomics

“…17−19 Methods to mitigate this effect have been proposed; these involve an additional MS3 scan to obtain quantitative reporter ions and would therefore affect the instrument duty cycle 20 or require specialized equipment. 21 A practical issue is the high cost of iTRAQ and TMT reagents for labeling milligrams of peptides, as is often required in phosphoproteomics 16,22 and studies of substoichiometric protein PTMs.…”

Properties of Probiotic Bacteria

“…In the application of DIGE, different samples are labeled with mass and charge matched spectrally resolvable fluorescent dyes and are then separated on the same 2-DE. After a number of cell divisions, each instance of this particular amino acid will be replaced by its isotope labeled analog (Oppermann et al, 2013). For instance, I report one simple DIGE procedure that can be usable by a simple laboratory.…”