Bhavin Patel1; Penny Jensen1; Aaron S. Gajadhar2; Sebastien Gallien3; Jae Choi1; Romain Huguet2; Graeme McAlister2; Derek Bailey2; Shannon Eliuk2; Markus Kellmann4; Tabiwang N. Arrey4; Alexander Harder4; Andreas Huhmer2; Kay Opperman1; John C Rogers1
ASMS 2019 Poster
Introduction
There is broad interest in quantifying dynamic protein phosphorylation states in cellular signaling pathways under different conditions. We have combined SMOAC (Sequential enrichment of Metal Oxide Affinity Chromatography), 146 AQUA™ heavy-labeled phosphopeptide standards, and internal standard triggered targeted MS to evaluate changes in phosphorylated protein abundance under different stimulation conditions. The specific phosphopeptides have been chosen to cover biologically interesting phosphosites from several different signaling pathways.
Methods
We developed an assay containing a pool of 146 AQUA heavy-labeled phosphopeptides from 89 signaling proteins. HeLa/A549 cells were grown with different stimulation conditions (hIGF-1/hEFG) before in-solution digestion. One milligram of each cell digest spiked with phosphopeptides standard was subjected to Thermo Scientific™ Hi-Select TiO2 phosphopeptide enrichment kit (PN#A32993). TiO2 flow-through/wash fractions were enriched with the Hi-Select Fe-NTA phophopeptide enrichment kit (PN#A32992). Both eluents were combined before LC-MS analysis using Thermo Scientific™ Dionex™ nanoLC™ system coupled to modified Orbitrap mass spectrometers. To ensure optimal measurement of each target, a novel targeting Thermo Scientific™ SureQuant™ method was performed where real-time heavy peptide detection triggered high-sensitivity measurement of endogenous targets. Data analysis was performed with Proteome Discoverer and Skyline software.
Preliminary Data
We have previously described our optimized SMOAC phosphopeptide enrichment method and we have shown with that method significant improvement in the number of phosphopeptides identified. In this study, we developed a targeted assay based upon 146 AQUA heavy-isotope phosphopeptide standards (96 serine, 26 threonine and 36 tyrosine modified peptides). More than 80% of peptides were quantified with SureQuant method. The phosphopeptide standards spiked into stimulated HeLa and A549 cell digest, followed by enrichment using the SMOAC method, allowed quantitation of endogenous phosphopeptides by a directed discovery (DDA with inclusion list and DIA) method. With an adapted internal standard triggered PRM method (SureQuant), using the modified Orbitrap MS instruments, we quantified multiple phosphopeptides in the SMOAC enriched HeLa and A549 stimulated digest. This SIL-triggered targeted analysis allowed much better quantitation of signaling pathway phosphorylated proteins by enhancing the detectability of targets and significantly improving measurement reproducibility across the different stimulation conditions. This targeted phosphopeptide assay coupled with SMOAC method and novel MS acquisition approach provided excellent quantitation, specificity and selectivity for signaling pathway analysis.
Novel Aspect
This phosphopeptide standard with novel targeted MS analysis allowed quantitation of phosphorylation changes from 89 signaling pathway proteins.
1Thermo Fisher Scientific, Rockford, IL
2Thermo Fisher Scientific, San Jose, CA
3Thermo Fisher Scientific, Precision Medicine Science Center, Cambridge, MA
4Thermo Fisher Scientific, Bremen, Germany
United States
