Top-down high-resolution electron capture dissociation mass spectrometry for comprehensive characterization of post-translational modifications in Rhesus monkey cardiac troponin I

Abstract

Top-down high-resolution electron capture dissociation (ECD) mass spectrometry (MS) is a powerful approach for comprehensive characterization of large proteins with labile post-translational modifications (PTMs). Phosphorylation of cardiac troponin I (cTnI) is a prominent determinant of cardiac function. Non-human primates (NHPs) are valuable models for biomedical research due to their genetic similarity to humans. To address the emerging need for in-depth understanding of NHP heart models, we have applied top-down high-resolution tandem mass spectrometry in conjugation with immunoaffinity chromatography purification to comprehensively characterize NHP cTnI. Our data revealed that cTnI affinity purified from NHP Rhesus monkey hearts was N-terminally acetylated and mono- or bis-phosphorylated. ECD unambiguously identified Ser22/Ser23 as the only basally phosphorylated sites with a phosphorylation order between these two sites (Ser23 phosphorylates prior to Ser22) which is different from those previously reported for mouse and human cTnI. Our study also strongly supports the nonergodic mechanism of ECD since no neutral loss of phosphates was observed in ECD spectra. Taken together, top-down MS with ECD is extremely valuable for studying labile PTMs in large proteins, which adds critical knowledge to our understanding of protein PTM regulations in health and disease.