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

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• 作者：Stephen F. Thoemmes ; Crystal A. Stutzke ; Yanmei Du ; Michael D. Browning ; Peter M. Buttrick ; Lori A. Walker
• 关键词：Phosphorylation ; Cardiac ; Troponin I ; Antibodies ; Cardiovascular disease ; Human
• 刊名：Journal of Immunological Methods
• 出版年：31 January, 2014
• 年：2014
• 卷：403
• 期：1-2
• 页码：66-71
• 全文大小：578 K

文摘

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-serine150 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 渭g 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.