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.

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

Figure 1

Representative hematoxylin and eosin stained sections of (A) normal mammary epithelium from a MTB−; TAN+ control and (B) a tumor from a MTB+; TAN+ mouse. (C) To highlight differences in the tyrosine phosphoproteome between MTB+; TAN+ tumor and normal MTB−; TAN+ mammary epithelium, tyrosine phosphoproteins were immunoprecipitated with the 4G10 antiphosphotyrosine antibody from 1 mg protein input, separated by 8% SDS-PAGE and detected by Western blot with 4G10. Arrows highlight prominent phosphoproteins not discernible in normal mammary epithelium.

Figure 2

Schematic of phosphotyrosine-enriched and total phosphoproteomic workflows. Phosphotyrosine enrichment was accomplished by immunoprecipitation with the PT66 antiphosphotyrosine antibody followed by PolyMAC-Ti in high recovery conditions. The total phosphoproteome was fractionated on a C18 column under neutral conditions followed by PolyMAC-Ti in high selectivity conditions.

Figure 3

Performance of PolyMAC-Ti with phosphotyrosine enrichment (A, C, and E) or RPLC fractionation at pH 8 for total phosphoproteome identification (B, D, and F). (A and B) The number of unique peptides detected at a 1% false discovery rate. (C and D) The number of unique phosphotyrosine, phosphoserine, and phosphothreonine sites detected. (E and F) The selectivity of phosphopeptide enrichment for each LC-MS/MS run from (A–D). Error bars represent 95% confidence intervals, and n is the total number of unique peptides within each run.

Figure 4

Linear motifs identified for phosphotyrosines, phosphoserines, and phosphothreonines. The motifs were simplified from motif-x analysis, available in the Supporting Information.

Figure 5

Connections between potential therapeutic targets for HER2-driven breast cancer. SFK: Src family kinases, which includes SRC, FYN, and LYN. Proteins shown in gray were observed with tyrosine phosphosites known to be activating. All shown inhibitors are FDA-approved or in clinical development, and were identified using DGIdb.