Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

Abstract

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction pathways and is tightly regulated by kinases and phosphatases. Therefore, characterizing the phosphoproteome may provide insights into identifying novel targets and biomarkers for oncologic therapy. Mass spectrometry provides a way to globally detect and quantify thousands of unique phosphorylation events. However, phosphopeptides are much less abundant than non-phosphopeptides, making biochemical analysis more challenging. To overcome this limitation, methods to enrich phosphopeptides prior to the mass spectrometry analysis are required. We describe a procedure to extract and digest proteins from tissue to yield peptides, followed by an enrichment for phosphotyrosine (pY) and phosphoserine/threonine (pST) peptides using an antibody-based and/or titanium dioxide (TiO2)-based enrichment method. After the sample preparation and mass spectrometry, we subsequently identify and quantify phosphopeptides using liquid chromatography-mass spectrometry and analysis software.

Targeting RET Kinase in Neuroendocrine Prostate Cancer

“…Cultured prostate cancer cells were scraped, pelleted, and snap frozen. Phosphopeptide enrichment and trypsin digestion were performed as described previously (16). Briefly, cells were lysed in 6 mol/L guanidium hydrochloride buffer (6 mol/L guanidinium chloride, 100 mmol/L Tris pH 8.5, 10 mmol/L Tris (2-carboxyethyl) phosphine, 40 mmol/L 2-chloroacetamide, 2 mmol/L Vanadate, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L Beta-glycerophosphate, 10 mg/mL N-octyl-glycoside), sonicated, and cleared.…”

Targeting RET Kinase in Neuroendocrine Prostate Cancer

“…Cultured prostate cancer cells were scraped, pelleted, and snap frozen. Phosphopeptide enrichment and trypsin digestion were performed as previously described (34). Briefly, cells were lysed in 6M guanidium hydrochloride buffer (6M Guanidinium chloride, 100mM Tris pH8.5, 10mM Tris (2-carboxyethyl) phosphine, 40mM 2-chloroacetamide, 2mM Vanadate, 2.5mM Sodium Pyrophosphate, 1mM Beta-glycerophosphate, 10 mg/ml N-octyl-glycoside), sonicated, and cleared.…”

“…Datasets are accessible through dataset identifiers PXD012970 and PXD012971 (37) through the ProteomeXchange Consortium via the PRIDE partner repository. We expanded upon our previously published mCRPC dataset by decreasing the phosphosite localization probability cutoff from 0.99 to 0.75 (34). This increased our identifications nearly 50% and have now reported those extra identifications in this manuscript as Supplemental Table 6.…”

Identification of clinical combination therapies to induce durable responses in kidney cancers

“…To obtain further insight into the signaling pathways acutely affected by dasatinib and cabozantinib co-treatment, we evaluated the changes in the phosphoproteome of the human NCCRCC cell line ACHN after dasatinib, cabozantinib and the combination treatment via quantitative, label-free quantitative mass spectrometry [32][33][34] .Supervised hierarchical clustering revealed duplicate samples clustered together, but that treatment altered phosphorylation levels of phosphopeptides with 3,369 phosphoserine and phosphothreonine (pST) peptides and 81 phosphotyrosine (pY) peptides significantly differed between treated and untreated cells (FDR <0.10, 0.20, respectively) ( Figure 4A). As expected based on known dasatinib and cabozantinib targets, the SRC family kinases (including SRC, Fyn, Hck, Lyn, and Fgr) and KDR (VEGF) were predicted to be significantly less active in the combination-treated cells based on pY kinase substrate enrichment analyses (KSEA) 33,35 ,an approach that estimates changes in a kinase's activity based on the collective phosphorylation changes of its identified substrates.…”

“…Phosphopeptides were enriched and analyzed by mass spectrometry as previously described in detail 32 .Briefly, cells were lysed and proteins extracted using a guanidinium-based lysis buffer.…”