Targeted Proteomics for Determining Phosphorylation Site-Specific Associations in Cardiovascular Disease
Posttranslational modifications (PTMs) are chemical or physical changes to proteins that can alter structure/function relationships and thus influence catalysis, cell localization, and interactions with other proteins and biomolecules. There are >200 recognized PTMs, generating a vast scope for altering protein function and increasing the potential for complex regulatory crosstalk between diverse modifications. Protein phosphorylation is of major significance because 30% to 50% of all cellular proteins may be targeted.1,2 Phosphorylation is a transient means of amplifying environmental signals to rapidly alter protein structure, interactions, and, ultimately, function. Although many studies have attempted to determine how aberrant signaling can contribute to the pathophysiology of diseases (eg, insulin stimulation3,4), very few have examined the associations of long-term alterations in protein phosphorylation during chronic disease. Within the myocyte, phosphorylation regulates numerous key functions.5 In this issue of Circulation, Zhang and colleagues6 have used targeted proteomics by multiple reaction monitoring (MRM) mass spectrometry (MS) to accurately quantify phosphorylated amino acids in cardiac troponin I (cTnI) associated with failing ischemic and idiopathic dilated cardiomyopathy hearts.
Article see p 1828
Traditionally, the study of single protein phosphorylation has relied on Western blot profiling with site-specific antibodies directed against known phosphorylation sites. Such an approach loses contextual information at the signal network and cellular level. This is critical, because the extent of protein phosphorylation mediates downstream consequences and can also aid in defining upstream regulation. Given the right conditions, including kinase activation, site accessibility, and adequate supply of phosphate (generally derived from ATP), nearly any serine, threonine, or tyrosine may be phosphorylated. The challenge therefore is 2-fold: adequately covering these residues analytically and determining those sites of modification that result in altered protein function. New approaches to determining the phospho-status of proteins aim to take an unbiased view, …