Plug-and-play analysis of the human phosphoproteome by targeted high-resolution mass spectrometry

Abstract

Systematic approaches to study cellular signaling require new phosphoproteomic techniques that reproducibly measure the same phosphopeptides across multiple replicates, conditions, and time points. Here we present a method to mine information from large-scale, heterogeneous phosphoproteomics datasets to rapidly generate robust targeted assays. We demonstrate the performance of our method by interrogating the IGF-1/AKT signaling pathway; and show that even rarely observed phosphorylation events can be consistently detected and precisely quantified.

Building ProteomeTools based on a complete synthetic human proteome

“…We recorded HCD spectra at six different normalized collision energies with the aim to identify conditions for the measurement of peptides by targeted assays such as SRM, PRM or SWATH 13, 14 . To evaluate if HCD spectra obtained in this study are suitable for this purpose, we compared our data to ~9,000 peptides from a SWATH spectral library built from proteome digests acquired on a QTOF instrument 15 .…”

Recent advances in phosphoproteomics and application to neurological diseases

“…133–135 For example, Lawrence and colleagues introduced a method to mine a large database of previous LC-MS analyses to assist with phosphopeptide selection and retention time scheduling; interestingly, for a set of 101 peptides, their “plug and play” PRM approach outperformed both DDA and DIA strategies in reproducibility and number of peptides identified. 136 Urisman et al . recently demonstrated that label-free PRM multiplexing could be expanded to quantify over 800 peptides from 150 kinases in a single analysis using the separation capabilities of a long monolithic silica-C18 capillary column.…”

Targeted mass spectrometry: An emerging powerful approach to unblock the bottleneck in phosphoproteomics

“…however, currently, targeted methods including PRM outperform DIA approaches in sensitivity and quantification accuracy [27,82].…”

“…The amount of novel information regarding phosphosites increases so rapidly that none of the classical techniques mentioned above can cope with the speed. Yet again, another MS-based strategy called targeted phosphoproteomics has emerged as a powerful resource to systematically monitor site-specific protein phosphorylation with high precision and sensitivity [27].…”