Use of protease proteomics to discover granzyme B substrates.
A wide variety of proteases play important roles in immunity, including the destruction of microbes, induction of apoptosis, antigen processing, and regulation of the immune response. Characterization of these proteases requires not only an understanding of substrate specificity, but also the identification of specific protein substrates. Recent advances in proteomics technology have introduced new techniques for the study of protease function. Here, we highlight a proteomic approach used in our laboratory that employs two-dimensional gel electrophoresis coupled with mass spectrometry to identify native protease substrates. With this technique, we have successfully detected both known and novel granzyme B substrates, characterized cleavage products, and identified a granzyme B cleavage site. This approach may serve as an important discovery tool for other immunologic proteases.
Bredemeyer
Andrew J
AJ
Division of Oncology, Department of Medicine, Siteman Cancer Center, Washington University School of Medicine, St. Louis, MO 63110, USA.
Townsend
R Reid
RR
Ley
Timothy J
TJ
D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional; D053804:Granzymes; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D051379:Mice; D010447:Peptide Hydrolases / Q000276:immunology* / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D012697:Serine Endopeptidases / Q000276:immunology* / Q000378:metabolism*; D013379:Substrate Specificity
D010447:Peptide Hydrolases; D053804:Granzymes; C509214:Gzmb protein, mouse; D012697:Serine Endopeptidases