There is broad interest in quantifying dynamic protein phosphorylation states in cellular signaling pathways under different conditions. Enrichment is necessary for better detection of the low abundant phosphorylated proteins, and multiplexed quantitation reagents parallelize processing across a multitude of experimental conditions. We have combined SMOAC (Sequential enrichment of Metal Oxide Affinity Chromatography), 146 AQUATM heavy-labeled phosphopeptide standards, and SureQuantTM 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.
HeLa/A549 cells were grown with different stimulation conditions (hIGF-1/hEFG) before in-solution digestion. One milligram of each digest spiked with phosphopeptides standard was subjected to SMOAC analysis using the Thermo Scientific Pierce Hi-SelectTM TiO2 and Fe_NTA phosphopeptide enrichment kits. Both eluents were combined before LC-MS analysis using Thermo Scientific Dionex nanoLC™ system coupled to Thermo Scientific™ Orbitrap Exploris™ 480 or Orbitrap Eclipse™ Tribrid™ Mass Spectrometers. To ensure optimal measurement of each target, a novel 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.
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. More than 90% of heavy peptides were quantified with high sensitivity and reproducibility across different MS acquisition methods. The phosphopeptide standards spiked into stimulated HeLa/A549 cell digest, followed by enrichment using the SMOAC method, allowed quantitation of about 60 endogenous phosphopeptides and 134 heavy phosphopeptides by PRM or SureQuant method.
This phosphopeptide standard with novel targeted MS analysis allowed quantitation of phosphorylation changes from >80 signaling pathway proteins.