A proteomic approach for the identification of cell-surface proteins shed by metalloproteases.
Proteolytic cleavage (shedding) of extracellular domains of many membrane proteins by metalloproteases is an important regulatory mechanism used by mammalian cells in response to environmental and physiological changes. Here we describe a proteomic system for analyzing cell surface shedding. The method utilized short-term culture supernatants from induced cells as starting material, followed by lectin-affinity purification, deglycosylation, and polyacrylamide gel electrophoresis separation. Relative quantitation of proteins was achieved via isotope dilution. In this study, a number of proteins already known to be shed were identified from activated monocytes and endothelial cells, thereby validating the method. In addition, a group of proteins were newly identified as being shed. The method provides an unbiased means to screen for shed proteins.
Guo
Lin
L
Immunex Corporation, Seattle, Washington 98101-2936, USA.
Eisenman
June R
JR
Mahimkar
Rajeev M
RM
Peschon
Jacques J
JJ
Paxton
Raymond J
RJ
Black
Roy A
RA
Johnson
Richard S
RS
D000328:Adult; D000478:Alkylation; D000818:Animals; D002273:Carcinogens / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002846:Chromatography, Affinity; D004229:Dithiothreitol / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004730:Endothelium, Vascular / Q000378:metabolism; D006023:Glycoproteins / Q000032:analysis; D006031:Glycosylation; D006720:Homozygote; D006801:Humans; D037102:Lectins / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000032:analysis*; D008666:Metalloendopeptidases / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D020543:Proteome; D012867:Skin / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002273:Carcinogens; D006023:Glycoproteins; D037102:Lectins; D008565:Membrane Proteins; D010446:Peptide Fragments; D020543:Proteome; D008666:Metalloendopeptidases; D013755:Tetradecanoylphorbol Acetate; D004229:Dithiothreitol