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Published on: June 13, 2014
Tissue phosphoproteomics with PolyMAC identifies potential therapeutic targets in a transgenic mouse model of HER2
Adam C Searleman1, Anton B Iliuk, Timothy S Collier
1Division of Oncology, Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA.
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.
Insights
Polymer-based metal ion affinity capture (PolyMAC) effectively enriches phosphopeptides from complex tissue samples. This method aids in identifying potential therapeutic targets for cancers like HER2-driven breast cancer.
Area of Science:
- Proteomics
- Cancer Biology
- Biochemistry
Background:
- Altered protein phosphorylation is a hallmark of human cancers and a target for therapy.
- Phosphopeptide enrichment is crucial for deep phosphoproteome analysis but challenging in complex tissues.
- Existing methods struggle with the complexity of tissue specimens.
Purpose of the Study:
- To evaluate polymer-based metal ion affinity capture (PolyMAC) for phosphoproteomic analysis of tissue specimens.
- To assess PolyMAC's efficacy in a HER2-driven breast cancer mouse model.
- To identify potential therapeutic targets using phosphoproteomic data.
Main Methods:
- Utilized polymer-based metal ion affinity capture (PolyMAC) for phosphopeptide enrichment.
- Applied phosphotyrosine immunoprecipitation combined with PolyMAC.
- Integrated reverse-phase liquid chromatography fractionation with PolyMAC.
- Performed LC-MS/MS analysis and linear motif analysis.
- Analyzed tyrosine phosphoproteome data using the Drug Gene Interaction database.
Main Results:
- Identified hundreds of unique phosphopeptides and multiple phosphosites (phosphotyrosine, phosphoserine, phosphothreonine).
- PolyMAC demonstrated high yield and specificity for phosphopeptide enrichment from tissue.
- Discovered a network of potential therapeutic targets, including Src family kinases, with existing inhibitors.
Conclusions:
- PolyMAC is a powerful and suitable technique for tissue phosphoproteomic analysis.
- The study identified potential therapeutic strategies for HER2-driven breast cancer.
- PolyMAC enhances the depth of phosphoproteome coverage in complex biological samples.
