title: Phosphorylation, but not alternative splicing or proteolytic degradation, is conserved in human and mouse cardiac troponin T.
abstract: Cardiac troponin T (cTnT), the tropomyosin binding subunit of the troponin complex, plays a pivotal regulatory role in the Ca(2+)-mediated interaction between actin thin filament and myosin thick filament. The post-translational modifications (PTMs) and alternative splicing of cTnT may represent important regulatory mechanisms of cardiac contractility. However, a complete characterization of PTMs and alternatively spliced isoforms in cTnT present in vivo is lacking. Top-down protein mass spectrometry (MS) analyzes whole proteins, thus providing a global view of all types of modifications, including PTMs and sequence variants, simultaneously in one spectrum without a priori knowledge. In this study, we applied an integrated immunoaffinity chromatography and top-down MS approach to comprehensively characterize PTMs and alternatively spliced isoforms of cTnT purified from healthy human and wild-type mouse heart tissue. High-resolution Fourier transform MS revealed that human cTnT (hcTnT) and mouse cTnT (mcTnT) have similar phosphorylation patterns, whereas higher molecular heterogeneity was observed for mcTnT than hcTnT. Further MS/MS fragmentation of monophosphorylated hcTnT and mcTnT by electron capture dissociation and collisionally activated dissociation unambiguously identified Ser1 as the conserved in vivo phosphorylation site. In contrast, we identified a single spliced isoform for hcTnT but three alternatively spliced isoforms for mcTnT. Moreover, we observed distinct proteolytic degradation products for hcTnT and mcTnT. This study also demonstrates the advantage of top-down MS/MS with complementary fragmentation techniques for the identification of modification sites in the highly acidic N-terminal region of cTnT.
authors.affiliation: Human Proteomics Program, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin 53706, USA.
authors.forename: Jiang
authors.initials: J
authors.lastname: Zhang
authors.forename: Han
authors.initials: H
authors.lastname: Zhang
authors.forename: Serife
authors.initials: S
authors.lastname: Ayaz-Guner
authors.forename: Yi-Chen
authors.initials: YC
authors.lastname: Chen
authors.forename: Xintong
authors.initials: X
authors.lastname: Dong
authors.forename: Qingge
authors.initials: Q
authors.lastname: Xu
authors.forename: Ying
authors.initials: Y
authors.lastname: Ge
mesh_terms: D000328:Adult; D017398:Alternative Splicing / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence / Q000235:genetics; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009200:Myocardial Contraction / Q000235:genetics; D009206:Myocardium / Q000201:enzymology / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism*; D010766:Phosphorylation / Q000235:genetics; D020033:Protein Isoforms / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000235:genetics; D020107:Troponin T / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D020033:Protein Isoforms; C545568:TNNT2 protein, human; D020107:Troponin T; D010447:Peptide Hydrolases