Determining Protease Substrates Within a Complex Protein Background Using the PROtein TOpography and Migration Analysis Platform (PROTOMAP).
The PROtein TOpography and Migration Analysis Platform (PROTOMAP) approach is a degradomics technique used to determine protease substrates within complex protein backgrounds. The method involves protein separation according to protein relative mobility, using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Gel lanes are then sliced into horizontal sections, and in-gel trypsin digestion performed for each gel slice. Extracted peptides and corresponding proteins are identified using liquid chromatography-tandem mass spectrometry and bioinformatics. Results are compiled in silico to generate a peptograph for every identified protein, being a pictorial representation of sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins shown by their peptograph to have migrated further through the gel (i.e., to a lower gel slice) in the lane containing the active protease(s) of interest, as compared to the control, are deemed putative protease substrates. PROTOMAP has broad applicability to a range of experimental conditions and protein pools. Coupling this with its simple and robust methodology, the PROTOMAP approach has emerged as a valuable tool with which to determine protease substrates in complex systems.
Australian Prostate Cancer Research Centre-Queensland, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia.|Translational Research Institute, Brisbane, Queensland, Australia.
R A
RA
Fuhrman-Luck
Translational Research Institute, Brisbane, Queensland, Australia.|Cancer Program, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia.
L M
LM
Silva
Protein Discovery Centre, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.
M L
ML
Hastie
Protein Discovery Centre, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.
J J
JJ
Gorman
Australian Prostate Cancer Research Centre-Queensland, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia. j.clements@qut.edu.au.|Translational Research Institute, Brisbane, Queensland, Australia. j.clements@qut.edu.au.
J A
JA
Clements
D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D010447:Peptide Hydrolases / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D012984:Software; D013223:Statistics as Topic / Q000379:methods; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry; D057188:Workflow
D010455:Peptides; D011506:Proteins; D010447:Peptide Hydrolases
Degradomics; PROTOMAP; PROtein TOpography and Migration Analysis Platform; Protease; Proteolysis; Proteomics; Substrate