Absolute quantification of cardiac troponin T by means of liquid chromatography/triple quadrupole tandem mass spectrometry.
A liquid chromatography/tandem mass spectrometric method for absolute quantification of cardiac troponin T (cTnT) in mouse heart tissue is presented. Even in such a complex biological sample, the multiple reaction monitoring acquisition mode allowed the selective and sensitive determination of a specific peptide, obtained by cTnT enzymatic digestion. The concentration of this cTnT-specific peptide was considered as a representation of the concentration of its parent protein. Quantification was carried out by means of the matrix-matched calibration curve, constructed by adding the synthetic standard of the target peptide and another synthetic structurally analogous peptide as internal standard. Method identification limit and method quantification limit were estimated as 60 and 110 ng of cTnT per mg of total extracted proteins, respectively. The developed label-free approach has been applied for the absolute quantitation of cTnT because of its diagnostic and prognostic value as cardiac disease marker. However, the method could be of general application, since it requires only the synthesis of two suitable peptides, a protein tryptic cleavage product and an internal standard.
Cavaliere
Chiara
C
Department of Chemistry, Sapienza University of Rome, Piazzale Aldo Moro 5, Rome, Italy.
Cucci
Francesca
F
Guarino
Chiara
C
Gubbiotti
Riccardo
R
Samperi
Roberto
R
Laganà
Aldo
A
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D032383:Myocytes, Cardiac / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D020107:Troponin T / Q000032:analysis*
D020107:Troponin T