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J Am Soc Mass Spectrom. 2018 Jun;29(6):1284-1294. doi: 10.1007/s13361-018-1925-y. Epub 2018 Apr 9.

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

Author information

1
Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, WI, 53705, USA.
2
Human Proteomics Program, University of Wisconsin-Madison, Madison, WI, 53705, USA.
3
Department of Cardiology, Shandong Provincial Hospital, Jinan, 250021, Shandong, People's Republic of China.
4
Institute of Computing Technology, Chinese Academy of Sciences, Beijing, 100190, People's Republic of China.
5
Department of Chemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA.
6
Department of Animal Science, University of Wisconsin-Madison, Madison, WI, 53706, USA.
7
Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, WI, 53705, USA. ge2@wisc.edu.
8
Human Proteomics Program, University of Wisconsin-Madison, Madison, WI, 53705, USA. ge2@wisc.edu.
9
Department of Chemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA. ge2@wisc.edu.

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 ᅟ.

KEYWORDS:

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

PMID:
29633223
PMCID:
PMC6109964
[Available on 2019-06-01]
DOI:
10.1007/s13361-018-1925-y

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