Label-Free Quantitation and Mapping of the ErbB2 Tumor Receptor by Multiple Protease Digestion with Data-Dependent (MS1) and Data-Independent (MS2) Acquisitions
The receptor tyrosine kinase ErbB2 is a breast cancer biomarker whose posttranslational modifications (PTMs) are a key indicator of its activation. Quantifying the expression and PTMs of biomarkers such as ErbB2 by selected reaction monitoring (SRM) mass spectrometry has several limitations, including minimal coverage and extensive assay development time. Therefore, we assessed the utility of two high resolution, full scan mass spectrometry approaches, MS1 Filtering and SWATH MS2, for targeted ErbB2 proteomics. Endogenous ErbB2 immunoprecipitated from SK-BR-3 cells was in-gel digested with trypsin, chymotrypsin, Asp-N, or trypsin plus Asp-N in triplicate. Data-dependent acquisition with an AB SCIEX TripleTOF 5600 and MS1 Filtering data processing was used to assess peptide and PTM coverage as well as the reproducibility of enzyme digestion. Data-independent acquisition (SWATH) was also performed for MS2 quantitation. MS1 Filtering and SWATH MS2 allow quantitation of all detected analytes after acquisition, enabling the use of multiple proteases for quantitative assessment of target proteins. Combining high resolution proteomics with multiprotease digestion enabled quantitative mapping of ErbB2 with excellent reproducibility, improved amino acid sequence and PTM coverage, and decreased assay development time compared to typical SRM assays. These results demonstrate that high resolution quantitative proteomic approaches are an effective tool for targeted biomarker quantitation. 1. Introduction Large-scale efforts to understand biological processes, such as functional genomics, systems biology, and cancer mutation analysis, continue to uncover master regulators of cell signaling and potential biomarkers of human disease [1–3]. Understanding the regulation of these biomarkers and validating their role in disease processes, however, depends on measurement of their expression and regulatory status in response to different cellular conditions, drug treatments, or patient samples. The receptor tyrosine kinase ErbB2 (HER2) is an important biomarker that is overexpressed in ~25% of all breast cancers, is a key drug target, and is a member of a biologically important family of tyrosine kinases. ErbB2 is known to be heavily regulated by posttranslational modifications (PTMs) which can modulate its kinase activity and protein-protein interaction partners [4–6]. ErbB2 is also subject to membrane-associated proteolytic processing and has several poorly understood isoform variants [7]. Mass spectrometry-based proteomics combined with stable-isotope labeling or tagging
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