Membrane protease degradomics: proteomic identification and quantification of cell surface protease substrates.
The modification of cell surface proteins by plasma membrane and soluble proteases is important for physiological and pathological processes. Methods to identify shed and soluble substrates are crucial to further define the substrate repertoire, termed the substrate degradome, of individual proteases. Identifying protease substrates is essential to elucidate protease function and involvement in different homeostatic and disease pathways. This characterisation is also crucial for drug target identification and validation, which would then allow the rational design of specific targeted inhibitors for therapeutic intervention. We describe two methods for identifying and quantifying shed cell surface protease targets in cultured cells utilising Isotope-Coded Affinity Tags (ICAT) and Isobaric Tags for Relative and Absolute Quantification (iTRAQ). As a model system to develop these techniques, we chose a cell-membrane expressed matrix metalloproteinase, MMP-14, but the concepts can be applied to proteases of other classes. By over-expression, or conversely inhibition, of a particular protease with careful selection of control conditions (e.g. vector or inactive protease) and differential labelling, shed proteins can be identified and quantified by mass spectrometry (MS), MS/MS fragmentation and database searching.
Butler
Georgina S
GS
Centre for Blood Research, University of British Columbia, Vancouver, BC, Canada.
Dean
Richard A
RA
Smith
Derek
D
Overall
Christopher M
CM
D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000737:chemistry* / Q000201:enzymology*; D002853:Chromatography, Liquid; D005260:Female; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry; D053511:Matrix Metalloproteinase 14 / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D055432:Metabolomics / Q000379:methods; D050505:Mutant Proteins / Q000378:metabolism; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D013379:Substrate Specificity; D014162:Transfection
D008565:Membrane Proteins; D050505:Mutant Proteins; D010447:Peptide Hydrolases; D053511:Matrix Metalloproteinase 14