Neonatal hearts respond to stress and function in an environment quite different from adult hearts. There is evidence that these functional differences not only reflect modifications in abundance and isoforms of sarcomeric proteins, but also in modulation of sarcomeric protein phosphorylation. Yet our understanding of changes in sarcomeric protein phosphorylation in development is incomplete. In experiments reported here, we first quantified intact sarcomeric protein phosphorylation status between neonatal and adult rat hearts employing comparative 2D-gel electrophoresis in conjunction with phospho-protein specific staining. Subsequently, we measured phosphorylation changes at the peptide level employing high resolution LTQ-FT mass spectrometry analysis of titanium dioxide enriched phosphor-peptides differentially labeled with ¹⁶O/¹⁸O during in-gel-digestion. We also employed Western blotting using phosphorylation-site-specific antibodies to measure phosphorylation changes. Our data demonstrate the novel finding that phosphorylation levels of myosin-binding protein C (MyBP-C) at Ser295 and Ser315, as well as tropomyosin Ser283, increased, whereas phosphorylation levels of MyBP-C Ser320 and myosin-light-chain-2 Ser15 decreased in neonatal hearts compared to the same sites in adult hearts. Although our data point up the significant challenges to understanding relations between protein phosphorylation and cardiac function, they do support the hypothesis that developmental changes in modulation of protein are functionally significant and correlate with the prevailing physiological state.