Proteomic identification of cellular protease substrates using isobaric tags for relative and absolute quantification (iTRAQ).
Identification of protease substrates is essential to identify and understand the functional consequences of normal and dysregulated proteolysis in disease on the proteome. Isobaric tags for relative and absolute quantification (iTRAQ) can be used to identify novel protease substrates in the cellular context. An amine-targeted iTRAQ tag labels tryptic peptides generated from the proteins and protease cleavage products of secreted proteins, as well as protein domains shed from the cell membrane or pericellular matrix of protease-transfected cells that have accumulated in conditioned medium; a second iTRAQ tag is used for control cells. MS/MS fragmentation enables sequencing of the pooled pairs of differently labeled but identical peptides and generates a low mass signature ion peak unique for each label. This signature ion peak identifies the peptides originating from the protease-transfected or control cells; comparison of the peak areas enables relative quantitation of the peptide between the samples.
Dean
Richard A
RA
University of British Columbia, Vancouver, British Columbia, Canada.
Smith
Derek
D
University of Victoria Proteomics Centre, Victoria, British Columbia, Canada.
Overall
Christopher M
CM
University of British Columbia, Vancouver, British Columbia, Canada.
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D017077:Culture Media, Conditioned; D008969:Molecular Sequence Data; D040901:Proteomics; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry
D017077:Culture Media, Conditioned; D014357:Trypsin