title: Characterization and validation of new tools for measuring site-specific cardiac troponin I phosphorylation.
abstract: Phosphorylation of cardiac troponin I is a well established mechanism by which cardiac contractility is modulated. However, there are a number of phosphorylation sites on TnI which contribute singly or in combination to influence cardiac function. Accordingly, methods for accurately measuring site-specific TnI phosphorylation are needed. Currently, two strategies are employed: mass spectrometry, which is costly, difficult and has a low throughput; and Western blotting using phospho-specific antibodies, which is limited by the availability of reagents. In this report, we describe a cohort of new site-specific TnI phosphoantibodies, generated against physiologically relevant phosphorylation sites, that are superior to the current commercially available antibodies: to phospho-serine 22/23 which shows a >5-fold phospho-specificity for phosphorylated TnI; to phospho-serine 43, which has >3-fold phospho-specificity for phosphorylated TnI; and phospho-serine 150 which has >2-fold phospho-specificity for phosphorylated TnI. These new antibodies demonstrated greater sensitivity and specificity for the phosphorylated TnI than the most widely used commercially available reagents. For example, at a protein load of 20 μg of total cardiac extract, a commercially available antibody recognized both phosphorylated and dephosphorylated TnI to the same degree. At the same protein load our phospho-serine 22/23 antibody exhibited no cross-reactivity with dephosphorylated TnI. These new tools should allow a more accurate assessment and a better understanding of the role of TnI phosphorylation in the response of the heart to pathologic stress.
authors.affiliation: University of Colorado Denver, Dept. of Medicine/Cardiology, Aurora, CO 80045, United States.
authors.forename: Stephen F
authors.initials: SF
authors.lastname: Thoemmes
authors.affiliation: PhosphoSolutions, Colorado Bioscience Park, 12635 East Montview Blvd., # 213, Aurora, CO 80045, United States.
authors.forename: Crystal A
authors.initials: CA
authors.lastname: Stutzke
authors.affiliation: University of Colorado Denver, Dept. of Medicine/Cardiology, Aurora, CO 80045, United States.
authors.forename: Yanmei
authors.initials: Y
authors.lastname: Du
authors.affiliation: PhosphoSolutions, Colorado Bioscience Park, 12635 East Montview Blvd., # 213, Aurora, CO 80045, United States.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Browning
authors.affiliation: University of Colorado Denver, Dept. of Medicine/Cardiology, Aurora, CO 80045, United States.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Buttrick
authors.affiliation: University of Colorado Denver, Dept. of Medicine/Cardiology, Aurora, CO 80045, United States. Electronic address: lori.walker@ucdenver.edu.
authors.forename: Lori A
authors.initials: LA
authors.lastname: Walker
mesh_terms: D000818:Animals; D000906:Antibodies; D000918:Antibody Specificity; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western / Q000379:methods*; D002417:Cattle; D004195:Disease Models, Animal; D006801:Humans; D051379:Mice; D009203:Myocardial Infarction / Q000276:immunology / Q000378:metabolism*; D009206:Myocardium / Q000276:immunology / Q000378:metabolism*; D010766:Phosphorylation; D011237:Predictive Value of Tests; D011499:Protein Processing, Post-Translational; D051381:Rats; D015203:Reproducibility of Results; D013552:Swine; D019210:Troponin I / Q000276:immunology / Q000378:metabolism*
chemical_list: D000906:Antibodies; D015415:Biomarkers; D019210:Troponin I
keywords: Antibodies; Cardiac; Cardiovascular disease; Human; Phosphorylation; Troponin I