title: Identification and Characterization of Cardiac Troponin T Fragments in Serum of Patients Suffering from Acute Myocardial Infarction.
abstract: BACKGROUND
  Cardiac troponin T (cTnT) is the preferred biomarker for the diagnosis of acute myocardial infarction (AMI). It has been suggested that cTnT is present predominantly in fragmented forms in human serum following AMI. In this study, we have used a targeted mass spectrometry assay and epitope mapping using Western blotting to confirm this hypothesis.
  
  
  METHODS
  cTnT was captured from the serum of 12 patients diagnosed with AMI using an immunoprecipitation technique employing the M11.7 catcher antibody and fractionated with SDS-PAGE. Coomassie-stained bands of 4 patients at 37, 29, and 16 kDa were excised from the gel, digested with trypsin, and analyzed on a Q Exactive instrument set on targeted Selected Ion Monitoring mode with data-dependent tandem mass spectrometry (MS/MS) for identification. Western blotting employing 3 different antibodies was used for epitope mapping.
  
  
  RESULTS
  Ten cTnT peptides of interest were targeted. By using MS/MS, all of these peptides were identified in the 37-kDa, intact, cTnT band. In the 29- and 16-kDa fragment bands, 8 and 4 cTnT-specific peptides were identified, respectively. Some of these peptides were "semitryptic," meaning that their C-termini were not formed by trypsin cleavage. The C-termini of these semitryptic peptides represent the C-terminal end of the cTnT molecules present in these bands. These results were confirmed independently by epitope mapping.
  
  
  CONCLUSIONS
  Using LC-MS, we have succeeded in positively identifying the 29- and 16-kDa fragment bands as cTnT-derived products. The amino acid sequences of the 29- and 16-kDa fragments are Ser79-Trp297 and Ser79-Gln199, respectively.
authors.affiliation: Department of Clinical Chemistry, Central Diagnostic Laboratory, Maastricht University Medical Centre, Maastricht, the Netherlands.
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Streng
authors.affiliation: Department of Clinical Chemistry, Central Diagnostic Laboratory, Maastricht University Medical Centre, Maastricht, the Netherlands.
authors.forename: Douwe
authors.initials: D
authors.lastname: de Boer
authors.affiliation: Department of Clinical Chemistry, Central Diagnostic Laboratory, Maastricht University Medical Centre, Maastricht, the Netherlands.
authors.forename: William P T M
authors.initials: WP
authors.lastname: van Doorn
authors.affiliation: Department of Human Biology, Maastricht University, Maastricht, the Netherlands.
authors.forename: Freek G
authors.initials: FG
authors.lastname: Bouwman
authors.affiliation: Department of Human Biology, Maastricht University, Maastricht, the Netherlands.
authors.forename: Edwin C M
authors.initials: EC
authors.lastname: Mariman
authors.affiliation: Department of Clinical Chemistry, Central Diagnostic Laboratory, Maastricht University Medical Centre, Maastricht, the Netherlands.
authors.forename: Otto
authors.initials: O
authors.lastname: Bekers
authors.affiliation: Department of Clinical Chemistry, Central Diagnostic Laboratory, Maastricht University Medical Centre, Maastricht, the Netherlands.
authors.forename: Marja P
authors.initials: MP
authors.lastname: van Dieijen-Visser
authors.affiliation: Department of Clinical Chemistry, Central Diagnostic Laboratory, Maastricht University Medical Centre, Maastricht, the Netherlands; will.wodzig@mumc.nl.
authors.forename: Will K W H
authors.initials: WK
authors.lastname: Wodzig
mesh_terms: D000208:Acute Disease; D015415:Biomarkers / Q000097:blood; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D009203:Myocardial Infarction / Q000097:blood* / Q000175:diagnosis; D053719:Tandem Mass Spectrometry; D020107:Troponin T / Q000097:blood*
chemical_list: D015415:Biomarkers; D020107:Troponin T