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Originally published In Press as doi:10.1074/mcp.M300028-MCP200 on May 23, 2003.
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Molecular & Cellular Proteomics 2:315-324, 2003.
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Isolation and Isotope Labeling of Cysteine- and Methionine-containing Tryptic Peptides

Application to the Study of Cell Surface Proteolysis

Min Shen, Lin Guo, Alison Wallace, Jeff Fitzner, June Eisenman, Erik Jacobson and Richard S. Johnson{ddagger}

From the Amgen Corporation, Seattle, Washington 98101-2936

Inexpensive methods were developed for isolating and isotopically labeling tryptic peptides that contain either cysteine or methionine. After covalently capturing cysteine-containing peptides with pyridyl disulfide reactive groups on agarose beads, extensive wash steps were applied, and the attached peptides were released using a reducing agent. This approach results in less nonspecifically bound peptides and eliminates the possibility of generating avidin peptide background ions that can arise when using methods based on biotin and avidin (e.g. isotope-coded affinity tag). The thiols were alkylated using either N-ethyl- or N-D5-ethyl-iodoacetamide, both of which can be synthesized in a single step using inexpensive reagents. This isotopic labeling does not greatly increase the peptide mass, nor does it affect the peptide ion charge state in electrospray ionization. In addition, methionine-containing peptides were captured using commercially available methionine-reactive beads, and relative quantitation of peptides was achieved by isotopic labeling of amino groups using activated esters of either nicotinic acid or D4-nicotinic acid. These methods were used to study the metalloprotease-mediated shedding of cell surface proteins from a mouse monocyte cell line that had been treated with a phorbol ester and lipopolysaccharide. In addition to the identification of proteins previously determined to be inducibly shed, three new shed proteins were identified: CD18, ICOS ligand, and tumor endothelial marker 7-related protein.

{ddagger} To whom correspondence should be addressed. Tel.: 206-265-6412; Fax: 206-621-5440; E-mail: jsrichar{at}amgen.com


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