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

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.

Integrating proteomics with electrochemistry for identifying kinase biomarkers

“…We show that discovery and detection of over-phosphorylated proteins and the relative kinases can be done in a relatively simple manner, which is very versatile. Methods for detection of phosphorylated proteins in tissues had been suggested before 36 – 38 and so were detection methods for specific kinases. 11 , 14 , 16 , 17 We combine the two aspects and show a direct path from the tissue level to the kinase detection stage.…”

“…The polyMAC has been applied to tissue phosphoproteomics in transgenic mouse model of HER2 positive breast cancer and identified potential therapeutic targets. 36 Phosphoproteomics has also been applied to directly identify kinase inhibitor targets in cancer cell lines and mouse xenografts. 43 Though these studies show the promise of phosphoproteomics to provide new targets for designing anticancer therapies, most current approaches are not accessible to individualized human tissue analysis yet.…”

“…HDGF 27 , 46 is a secreted protein 46 , 47 that stimulates growth of several cell types including smooth muscle cells, 48 endothelial cells 49 and lung epithelial cells. 20 , 21 , 36 , 51 HDGF was previously linked to lung cancer and specifically to poor clinical outcome. It was shown to cause tumorigenesis in vivo .…”

Comparing Multistep Immobilized Metal Affinity Chromatography and Multistep TiO2 Methods for Phosphopeptide Enrichment

“…There are several approaches for enriching phosphopeptides from the peptide mixtures, including immobilized metal affinity chromatography (IMAC) with Fe 3+ or Ti 4+ 7,8,9 , metal oxide affinity chromatography (MOAC) with TiO 2 10,11 , and immuno-affinity based methods 12 . Other methods such as polymer-based metal ion affinity capture (PolyMAC) 13,14 and polymer-oxotitanium-modified gold wafer (Au-P-oxoTi) 15 result in successful phosphopeptide enrichment.…”

Recent advances in phosphoproteomics and application to neurological diseases

“…59, 60 This strategy has demonstrated outstanding selectivity, sensitivity, and recovery when compared to Fe-IMAC and TiO 2 for complex samples, and this strategy has been applied to study the phosphoproteome of animals and plants. 61–64 …”