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Comprehensive Characterization of Swine Cardiac Troponin T Proteoforms by Top-Down Mass Spectrometry

  • Ziqing Lin
  • Fang Guo
  • Zachery R. Gregorich
  • Ruixiang Sun
  • Han Zhang
  • Yang Hu
  • Dhanansayan Shanmuganayagam
  • Ying Ge
Focus: Mass Spectrometry in Glycobiology and Related Fields: Research Article

Abstract

Cardiac troponin T (cTnT) regulates the Ca2+-mediated interaction between myosin thick filaments and actin thin filaments during cardiac contraction and relaxation. cTnT is released into the blood following injury, and increased serum levels of the protein are used clinically as a biomarker for myocardial infarction. Moreover, mutations in cTnT are causative in a number of familial cardiomyopathies. With the increasing use of large animal (swine) model to recapitulate human diseases, it is essential to characterize species-dependent protein sequence variants, alternative RNA splicing, and post-translational modifications (PTMs), but challenges remain due to the incomplete database and lack of validation of the predicted splicing isoforms. Herein, we integrated top-down mass spectrometry (MS) with online liquid chromatography (LC) and immunoaffinity purification to comprehensively characterize miniature swine cTnT proteoforms, including those arising from alternative RNA splicing and PTMs. A total of seven alternative splicing isoforms of cTnT were identified by LC/MS from swine left ventricular tissue, with each isoform containing un-phosphorylated and mono-phosphorylated proteoforms. The phosphorylation site was localized to Ser1 for the mono-phosphorylated proteoforms of cTnT1, 3, 4, and 6 by online MS/MS combining collisionally activated dissociation (CAD) and electron transfer dissociation (ETD). Offline MS/MS on Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer with CAD and electron capture dissociation (ECD) was then utilized to achieve deep sequencing of mono-phosphorylated cTnT1 (35.2 kDa) with a high sequence coverage of 87%. Taken together, this study demonstrated the unique advantage of top-down MS in the comprehensive characterization of protein alternative splicing isoforms together with PTMs.

Graphical Abstract

Keywords

Cardiac troponin Heart disease Proteoform Top-down proteomics Collisionally activated dissociation Electron-transfer dissociation Electron-capture dissociation 

Notes

Acknowledgements

This work is dedicated to Professor Catherine E. Costello, the recipient of the 2017 Award for a Distinguished Contribution in Mass Spectrometry for her pioneering contributions to the development of tandem mass spectrometry of glycans and glycoconjugates. We would like to thank Bifan Chen for critical reading of this manuscript.

Funding Information

Financial support was kindly provided by NIH R01 HL109810 and HL096971 (to Y.G.). Y. G. would like to acknowledge NIH R01 GM117058 and S10 OD018475.

Compliance with Ethical Standards

All procedures were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals with protocols approved by the Animal Care and Use Committee of University of Wisconsin.

Supplementary material

13361_2018_1925_MOESM1_ESM.docx (1.7 mb)
ESM 1 (DOCX 1770 kb)

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Copyright information

© American Society for Mass Spectrometry 2018

Authors and Affiliations

  • Ziqing Lin
    • 1
    • 2
  • Fang Guo
    • 1
    • 3
  • Zachery R. Gregorich
    • 1
  • Ruixiang Sun
    • 1
    • 4
  • Han Zhang
    • 5
  • Yang Hu
    • 1
  • Dhanansayan Shanmuganayagam
    • 6
  • Ying Ge
    • 1
    • 2
    • 5
  1. 1.Department of Cell and Regenerative BiologyUniversity of Wisconsin-MadisonMadisonUSA
  2. 2.Human Proteomics ProgramUniversity of Wisconsin-MadisonMadisonUSA
  3. 3.Department of CardiologyShandong Provincial HospitalJinanPeople’s Republic of China
  4. 4.Institute of Computing TechnologyChinese Academy of SciencesBeijingPeople’s Republic of China
  5. 5.Department of ChemistryUniversity of Wisconsin-MadisonMadisonUSA
  6. 6.Department of Animal ScienceUniversity of Wisconsin-MadisonMadisonUSA

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