Tissue phosphoproteomics with PolyMAC identifies potential therapeutic targets in a transgenic mouse model of HER2 positive breast cancer

Electrophoresis. 2014 Dec;35(24):3463-9. doi: 10.1002/elps.201400022. Epub 2014 Jun 12.

Abstract

Altered protein phosphorylation is a feature of many human cancers that can be targeted therapeutically. Phosphopeptide enrichment is a critical step for maximizing the depth of phosphoproteome coverage by MS, but remains challenging for tissue specimens because of their high complexity. We describe the first analysis of a tissue phosphoproteome using polymer-based metal ion affinity capture (PolyMAC), a nanopolymer that has excellent yield and specificity for phosphopeptide enrichment, on a transgenic mouse model of HER2-driven breast cancer. By combining phosphotyrosine immunoprecipitation with PolyMAC, 411 unique peptides with 139 phosphotyrosine, 45 phosphoserine, and 29 phosphothreonine sites were identified from five LC-MS/MS runs. Combining reverse phase liquid chromatography fractionation at pH 8.0 with PolyMAC identified 1571 unique peptides with 1279 phosphoserine, 213 phosphothreonine, and 21 phosphotyrosine sites from eight LC-MS/MS runs. Linear motif analysis indicated that many of the phosphosites correspond to well-known phosphorylation motifs. Analysis of the tyrosine phosphoproteome with the Drug Gene Interaction database uncovered a network of potential therapeutic targets centered on Src family kinases with inhibitors that are either FDA-approved or in clinical development. These results demonstrate that PolyMAC is well suited for phosphoproteomic analysis of tissue specimens.

Keywords: Breast Cancer; Drug identification; HER2; Phosphoproteomics; PolyMAC.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Chromatography, Affinity / methods
  • Female
  • Male
  • Mammary Neoplasms, Experimental / chemistry
  • Mammary Neoplasms, Experimental / enzymology
  • Mammary Neoplasms, Experimental / metabolism*
  • Mice
  • Mice, Transgenic
  • Phosphoamino Acids / analysis*
  • Phosphoamino Acids / metabolism
  • Phosphopeptides / analysis*
  • Phosphopeptides / chemistry
  • Phosphopeptides / metabolism
  • Proteomics / methods*
  • Receptor, ErbB-2 / biosynthesis
  • Tandem Mass Spectrometry
  • Tissue Array Analysis / methods*

Substances

  • Phosphoamino Acids
  • Phosphopeptides
  • Erbb2 protein, mouse
  • Receptor, ErbB-2