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Submitted on October 30, 2001
Protein Chemistry Department, Genentech, Inc., South San Francisco, CA 94080
Corresponding Author: arnott{at}gene.com
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 compared between samples. Membrane protein extracts were labeled with custom isotope-coded affinity tag (ICAT) reagents, digested, and the labeled peptides 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 ICAT-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 PSCA and ErbB2 were detected in prostate and breast tumor cell lines in which they are expressed in known abundances spanning orders of magnitude.
Revised on December 18, 2001
Accepted on December 19, 2001
Selective detection of membrane proteins without antibodies: A mass spectrometric version of the western blot
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