Strategy for analysis of cardiac troponins in biological samples with a combination of affinity chromatography and mass spectrometry

Ralf Labugger, Jeremy A. Simpson, Michelle Quick, Heather A. Brown, Christine E. Collier, Irina Neverova, Jennifer E. Van Eyk

Research output: Contribution to journalArticle

Abstract

Background: Cardiac troponins are modified during ischemic injury and are found as a heterogeneous mixture in blood of patients with cardiovascular diseases. We present a strategy to isolate cardiac troponins from human biological material, by use of affinity chromatography, and to provide samples ready for direct analysis by mass spectrometry. Methods: Cardiac troponins were isolated from human left ventricular tissue by affinity chromatography. Isolated troponins were either eluted and analyzed by Western blot or enzymatically digested while bound to affinity beads. The resulting peptide mixture was subjected to mass spectrometry for protein identification and characterization. The same method was used to analyze serum from patients with acute myocardial infarction (AMI). Results: Affinity chromatography with antibodies specific for one cardiac troponin subunit facilitated the isolation of the entire cardiac troponin complex from myocardial tissue. The three different proteases used for enzymatic digestion increased the total protein amino acid sequence coverage by mass spectrometry for the three cardiac troponin subunits. Combined amino acid sequence coverages for cardiac troponin I, T, and C (cTnI, cTnT, cTnC) were 54%, 48%, and 40%, respectively. To simulate matrix effects on the affinity chromatography - mass spectrometry approach, we diluted tissue homogenate in cardiac troponin-free serum. Sequence coverages in this case were 44%, 41%, and 19%, respectively. Finally, affinity chromatography - mass spectrometry analysis of AMI serum revealed the presence of cardiac troponins in a wide variety of its free and/or complexed subunits, including the binary cTnI-cTnC and cTnI-cTnC-cTnT complexes. Conclusions: Affinity chromatography - mass spectrometry allows the extraction and analysis of cardiac troponins from biological samples in their natural forms. We were, for the first time, able to directly confirm the presence of cardiac troponin complexes in human serum after AMI. This approach could assist in more personalized risk stratification as well as the search for reference materials for cardiac troponin diagnostics.

Original languageEnglish (US)
Pages (from-to)873-879
Number of pages7
JournalClinical Chemistry
Volume49
Issue number6
DOIs
StatePublished - Jun 1 2003
Externally publishedYes

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Affinity chromatography
Troponin
Affinity Chromatography
Mass spectrometry
Mass Spectrometry
Myocardial Infarction
Tissue
Serum
Amino Acid Sequence
Troponin C
Amino Acids
Troponin T
Troponin I
Biological materials
Digestion
Proteins
Blood
Peptide Hydrolases
Cardiovascular Diseases

ASJC Scopus subject areas

  • Clinical Biochemistry

Cite this

Labugger, R., Simpson, J. A., Quick, M., Brown, H. A., Collier, C. E., Neverova, I., & Van Eyk, J. E. (2003). Strategy for analysis of cardiac troponins in biological samples with a combination of affinity chromatography and mass spectrometry. Clinical Chemistry, 49(6), 873-879. https://doi.org/10.1373/49.6.873

Strategy for analysis of cardiac troponins in biological samples with a combination of affinity chromatography and mass spectrometry. / Labugger, Ralf; Simpson, Jeremy A.; Quick, Michelle; Brown, Heather A.; Collier, Christine E.; Neverova, Irina; Van Eyk, Jennifer E.

In: Clinical Chemistry, Vol. 49, No. 6, 01.06.2003, p. 873-879.

Research output: Contribution to journalArticle

Labugger, R, Simpson, JA, Quick, M, Brown, HA, Collier, CE, Neverova, I & Van Eyk, JE 2003, 'Strategy for analysis of cardiac troponins in biological samples with a combination of affinity chromatography and mass spectrometry', Clinical Chemistry, vol. 49, no. 6, pp. 873-879. https://doi.org/10.1373/49.6.873
Labugger, Ralf ; Simpson, Jeremy A. ; Quick, Michelle ; Brown, Heather A. ; Collier, Christine E. ; Neverova, Irina ; Van Eyk, Jennifer E. / Strategy for analysis of cardiac troponins in biological samples with a combination of affinity chromatography and mass spectrometry. In: Clinical Chemistry. 2003 ; Vol. 49, No. 6. pp. 873-879.
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AU - Quick, Michelle

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AU - Collier, Christine E.

AU - Neverova, Irina

AU - Van Eyk, Jennifer E.

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