Phosphopeptide enrichment coupled with label-free quantitative mass spectrometry to investigate the phosphoproteome in prostate cancer

Larry C. Cheng, Zhen Li, Thomas G. Graeber, Nicholas A. Graham, Justin M Drake

Research output: Contribution to journalArticle

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 (TiO 2 )-based enrichment method. After the sample preparation and mass spectrometry, we subsequently identify and quantify phosphopeptides using liquid chromatography-mass spectrometry and analysis software.

Original languageEnglish (US)
Article numbere57996
JournalJournal of Visualized Experiments
Volume2018
Issue number138
DOIs
StatePublished - Aug 2 2018
Externally publishedYes

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Phosphopeptides
Mass spectrometry
Labels
Mass Spectrometry
Prostatic Neoplasms
Phosphorylation
Proteins
Peptides
Phosphoserine
Signal transduction
Phosphotyrosine
Phosphatases
Liquid chromatography
Biomarkers
Threonine
Phosphoric Monoester Hydrolases
Antibodies
Liquid Chromatography
Titanium dioxide
Signal Transduction

Keywords

  • Cancer research
  • Cell signaling
  • Issue 138
  • Kinases
  • Mass spectrometry
  • Phosphoproteomics
  • Phosphorylation
  • Prostate cancer
  • Proteomics

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Video-Audio Media

Cite this

Phosphopeptide enrichment coupled with label-free quantitative mass spectrometry to investigate the phosphoproteome in prostate cancer. / Cheng, Larry C.; Li, Zhen; Graeber, Thomas G.; Graham, Nicholas A.; Drake, Justin M.

In: Journal of Visualized Experiments, Vol. 2018, No. 138, e57996, 02.08.2018.

Research output: Contribution to journalArticle

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AU - Drake, Justin M

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