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Originally published In Press as doi:10.1074/mcp.M100027-MCP200 on December 19, 2001.
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Molecular & Cellular Proteomics 1:148-156, 2002.
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Selective Detection of Membrane Proteins Without Antibodies

A Mass Spectrometric Version of the Western Blot*

David Arnott{ddagger},§, Adrianne Kishiyama{ddagger}, Elizabeth A. Luis{ddagger}, Sarah G. Ludlum{ddagger}, James C. Marsters, Jr. and John T. Stults{ddagger}

{ddagger} Department of Protein Chemistry, Genentech, Inc., South San Francisco, California 94080
Department of BioOrganic Chemistry, Genentech, Inc., South San Francisco, California 94080

A method has been developed, called the mass western experiment in analogy to the Western blot, to detect the presence of specific proteins in complex mixtures without the need for antibodies. Proteins are identified with high sensitivity and selectivity, and their abundances are compared between samples. Membrane protein extracts were labeled with custom isotope-coded affinity tag reagents and digested, and the labeled peptides were analyzed by liquid chromatography-tandem mass spectrometry. Ions corresponding to anticipated tryptic peptides from the proteins of interest were continuously subjected to collision-induced dissociation in an ion trap mass spectrometer; heavy and light isotope-coded affinity tag-labeled peptides were simultaneously trapped and fragmented accomplishing identification and quantitation in a single mass spectrum. This application of ion trap selective reaction monitoring maximizes sensitivity, enabling analysis of peptides that would otherwise go undetected. The cell surface proteins prostate stem cell antigen (PSCA) and ErbB2 were detected in prostate and breast tumor cell lines in which they are expressed in known abundances spanning orders of magnitude.

§ To whom correspondence should be addressed: Genentech, Inc., 1 DNA Way, MS #63, South San Francisco, CA 94080. Tel.: 650-225-1240; Fax: 650-225-5945; E-mail: arnott{at}gene.com.


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