Deciphering modifications in swine cardiac troponin I by top-down high-resolution tandem mass spectrometry.
Cardiac troponin I (cTnI) is an important regulatory protein in cardiac muscle, and its modification represents a key mechanism in the regulation of cardiac muscle contraction and relaxation. cTnI is often referred to as the "gold-standard" serum biomarker for diagnosing patients with acute cardiac injury since it is unique to the heart and released into the circulation following necrotic death of cardiac tissue. The swine (Sus scrofa) heart model is extremely valuable for cardiovascular research since the heart anatomy and coronary artery distribution of swine are almost identical to those of humans. Herein, we report a comprehensive characterization of the modifications in swine cTnI using top-down high-resolution tandem mass spectrometry in conjugation with immunoaffinity chromatography purification. High-resolution high accuracy mass spectrometry revealed that swine cTnI affinity purified from domestic pig hearts was N-terminally acetylated and phosphorylated. Electron capture disassociation is uniquely suited for localization of labile phosphorylations, which unambiguously identified Ser22/Ser23 as the only basally phosphorylated sites that are well-known to be regulated by protein kinase A and protein kinase C. Moreover, a combination of tandem mass spectrometry with sequence homology alignment effectively localized a single amino acid polymorphism, V116A, representing a novel genetic variant of swine cTnI. Overall, our studies demonstrated the unique power of top-down high-resolution tandem mass spectrometry in the characterization of protein modifications, including labile phosphorylation and unexpected sequence variants.
Zhang
Jiang
J
Human Proteomics Program, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Dong
Xintong
X
Hacker
Timothy A
TA
Ge
Ying
Y
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D008969:Molecular Sequence Data; D009206:Myocardium / Q000737:chemistry* / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D016415:Sequence Alignment; D013552:Swine; D053719:Tandem Mass Spectrometry / Q000379:methods*; D019210:Troponin I / Q000737:chemistry* / Q000378:metabolism
D019210:Troponin I