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.
Bibliographical noteFunding Information:
We thank members of the Drake lab for providing advice and input on the manuscript. We also thank the members of the Biological Mass Spectrometry Facility of Robert Wood Johnson Medical School and Rutgers, The State University of New Jersey, for providing advice and performing mass spectrometry on our samples. Larry C. Cheng is supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number T32 GM008339. Thomas G. Graeber is supported by the NCI/NIH (SPORE in Prostate Cancer P50 CA092131; P01 CA168585) and an American Cancer Society Research Scholar Award (RSG-12-257-01-TBE). Justin M. Drake is supported by the Department of Defense Prostate Cancer Research Program W81XWH-15-1-0236, Prostate Cancer Foundation Young Investigator Award, the New Jersey Health Foundation, and a Precision Medicine Initiative Pilot Award from the Rutgers Cancer Institute of New Jersey.
© 2018 Journal of Visualized Experiments.
- Cancer research
- Cell signaling
- Issue 138
- Mass spectrometry
- Prostate cancer