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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.

Original publication

DOI

10.1016/j.jim.2013.11.020

Type

Journal article

Journal

J Immunol Methods

Publication Date

31/01/2014

Volume

403

Pages

66 - 71

Keywords

Antibodies, Cardiac, Cardiovascular disease, Human, Phosphorylation, Troponin I, Animals, Antibodies, Antibody Specificity, Biomarkers, Blotting, Western, Cattle, Disease Models, Animal, Humans, Mice, Myocardial Infarction, Myocardium, Phosphorylation, Predictive Value of Tests, Protein Processing, Post-Translational, Rats, Reproducibility of Results, Swine, Troponin I