Optimization of cardiac troponin I pull-down by IDM affinity beads and SELDI.
Cardiac troponin I (cTnI) is a key regulator of cardiac muscle contraction. Upon myocardial cell injury, cTnI is lost from the cardiac myocyte and can be detected in serum, in some cases with specific disease-induced modifications, making it an important diagnostic marker for acute myocardial injury. Presently, hospital laboratories use enzyme-linked immunosorbent assays to detect cTnI, but this type of analysis lacks information about modified forms of protein (degradation or phosphorylation) that may give a more specific diagnosis from either serum or biopsies. Because cardiac and serum tissues are widely used for proteomic analysis, it is important to detect these cTnI posttranslational modifications. Therefore, we have chosen to optimize the enrichment and detection of cTnI protein by IDM Affinity Bead pull-down and surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS or SELDI) analyses. By adjusting the chemical compositions of the buffers, we have retained antibody specificity and enriched for different forms of cTnI and its associated proteins.
Bovenkamp
Diane E
DE
Department of Medicine, Johns Hopkins University, Baltimore, MD, USA.
Stanley
Brian A
BA
Van Eyk
Jennifer E
JE
D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D051379:Mice; D009206:Myocardium / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D019210:Troponin I / Q000032:analysis* / Q000378:metabolism
D019210:Troponin I