Table of Contents
International Journal of Proteomics
Volume 2013, Article ID 791985, 11 pages
http://dx.doi.org/10.1155/2013/791985
Research Article

Label-Free Quantitation and Mapping of the ErbB2 Tumor Receptor by Multiple Protease Digestion with Data-Dependent (MS1) and Data-Independent (MS2) Acquisitions

1The Buck Institute for Research on Aging, 8001 Redwood Boulevard, Novato, CA 94945, USA
2Department of Medicine and Division of Oncology-Hematology, University of California, San Francisco, CA 94143, USA
3Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143, USA

Received 14 September 2012; Accepted 6 February 2013

Academic Editor: Mu Wang

Copyright © 2013 Jason M. Held et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

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.