Strategy for analysis of cardiac troponins in biological samples with a combination of affinity chromatography and mass spectrometry.
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 coverage for cardiac troponin I, T, and C (cTnI, cTnT, cTnC) was 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 coverage in this case was 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.
Labugger
Ralf
R
Department of Physiology, Queen's University, Kingston, Ontario, K7L 3N6 Canada.
Simpson
Jeremy A
JA
Quick
Michelle
M
Brown
Heather A
HA
Collier
Christine E
CE
Neverova
Irina
I
Van Eyk
Jennifer E
JE
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009203:Myocardial Infarction / Q000175:diagnosis; D009206:Myocardium / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019209:Troponin C / Q000097:blood* / Q000302:isolation & purification; D019210:Troponin I / Q000097:blood* / Q000302:isolation & purification; D020107:Troponin T / Q000097:blood* / Q000302:isolation & purification
D019209:Troponin C; D019210:Troponin I; D020107:Troponin T