Proteomic analysis of pharmacologically preconditioned cardiomyocytes reveals novel phosphorylation of myosin light chain 1

Proteomic analysis of rabbit ventricular myocytes revealed a novel posttran slational modification to myosin light chain I (MLCl), consisting of phosph orylation at two sites. Subproteomic extraction to isolate myofilament-enri ched fractions enabled determination of the extent of phosphorylation, whic h increased from 25.7 +/- 1.6% to 34.0 +/- 2.7% (mean +/- SE. n=4; P < 0.05 ) after adenosine treatment at levels sufficient to pharmacologically preco ndition the myocytes (100 mu mol/L). Mass spectrometry of MLCl tryptic dige sts identified two peptide fragments modified by phosphorylation. These two phosphopeptides were characterized by peptide mass fingerprinting to deter mine the phosphorylation sites within rabbit ventricular MLCl, which corres pond to Thr69 and Ser200 of rat MLCl, and to Thr64 and Ser194 or 195 of hum an MLCl. This proteomic analysis of preconditioned myocardium has revealed a previously unsuspected in vivo posttranslational modification to MLCl.