A highly multiplexed quantitative phosphosite assay for biology and preclinical studies

Abstract

Reliable methods to quantify dynamic signaling changes across diverse pathways are needed to better understand the effects of disease and drug treatment in cells and tissues but are presently lacking. Here, we present SigPath, a targeted mass spectrometry (MS) assay that measures 284 phosphosites in 200 phosphoproteins of biological interest. SigPath probes a broad swath of signaling biology with high throughput and quantitative precision. We applied the assay to investigate changes in phospho‐signaling in drug‐treated cancer cell lines, breast cancer preclinical models, and human medulloblastoma tumors. In addition to validating previous findings, SigPath detected and quantified a large number of differentially regulated phosphosites newly associated with disease models and human tumors at baseline or with drug perturbation. Our results highlight the potential of SigPath to monitor phosphoproteomic signaling events and to nominate mechanistic hypotheses regarding oncogenesis, response, and resistance to therapy.

Rapid and In-Depth Coverage of the (Phospho-)Proteome With Deep Libraries and Optimal Window Design for dia-PASEF

“…We first analyzed the resulting files with DIA-NN against our deep phospho-library. In agreement with our simulations, the original dia-PASEF method identified 8,199 phosphosites and 13,485 phosphopeptides whereas the optimal method detected 28% more phosphosites (10,510) and 43% more phosphorylated peptides (19,258) (Fig. 5E).…”

Quantitative Cell Proteomic Atlas: Pathway-scale targeted mass spectrometry for high-resolution functional profiling of cell signaling

“…QCPA peptides were synthesized by JPT Peptide Technologies GmbH (Berlin, Germany) as SpikeMix Peptide Pools (purity>75%), in sets of 96 peptides per pool. 13 C6 15 N2-lysine and 13 C6 15 N4-arginine residues were incorporated as Cterminal amino acids. All C residues were carbamidomethylated.…”

“…For example, biological functions of protein phosphorylation have been documented extensively, including causal linkage of aberrant activity of protein kinases and phosphatases to cancer onset and progression [10][11][12]. Systematic quantification of specifically phosphorylated regulatory protein domains has been used to determine cellular functional states [13] and to identify molecular mechanisms linked to disease progression, and response and resistance to therapy [13][14][15][16][17][18]. To aid the detection of phosphorylated proteins and peptides, several immune-and metal affinity enrichment techniques have been recently developed [19][20][21][22].…”

Modulation of huntingtin degradation by cAMP-dependent protein kinase A (PKA) phosphorylation of C-HEAT domain Ser2550

“…Following successful labeling reactions were quenched and samples for each plex were mixed and desalted. Resulting sample for each plex was first enriched by phosphotyrosine containing peptides using P1000 anti-phosphotyrosine antibody (CST) and enriched fraction was analyzed by LC-MS/MS (Keshishian et al, 2021). Flow through of the enrichment was desalted and fractionated on a 3.5 μm Agilent Zorbax 300 Extend-C18 column (4.6 mm ID x 250 mm length) into 24 fractions.…”