Characterization and validation of new tools for measuring site-specific cardiac troponin I phosphorylation

J Immunol Methods. 2014 Jan 31;403(1-2):66-71. doi: 10.1016/j.jim.2013.11.020. Epub 2013 Nov 26.


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.

Keywords: Antibodies; Cardiac; Cardiovascular disease; Human; Phosphorylation; Troponin I.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Validation Study

MeSH terms

  • Animals
  • Antibodies*
  • Antibody Specificity
  • Biomarkers / metabolism
  • Blotting, Western / methods*
  • Cattle
  • Disease Models, Animal
  • Humans
  • Mice
  • Myocardial Infarction / immunology
  • Myocardial Infarction / metabolism*
  • Myocardium / immunology
  • Myocardium / metabolism*
  • Phosphorylation
  • Predictive Value of Tests
  • Protein Processing, Post-Translational*
  • Rats
  • Reproducibility of Results
  • Swine
  • Troponin I / immunology
  • Troponin I / metabolism*


  • Antibodies
  • Biomarkers
  • Troponin I