MyBP-C slow is phosphorylated by PKA and PKC in vitro. A: Schematic representation of the same “hypothetical” protein shown in Fig. 1A, highlighting recombinant MyBP-C slow constructs. Recombinant polypeptides are denoted as GST-NH2 terminal construct, GST-C7 construct and GST-COOH terminal construct and detected by SYPRO ruby total protein stain (B). C–F: Phosphorylation events mediated by PKA and PKC were identified in the GST-NH2-terminal construct via mass spectrometry analysis using LTQ-orbitrap. MS/MS scans of peptides carrying phosphorylated Ser-59 by PKA (C), Ser-62 by PKA (D) and Ser-204 by PKA (E) and PKC (F), identified by Bioworks-SEQUEST, with Xcorr scores of 4.471, 4.757, 4.142 and 4.475, respectively, and manually validated are shown.