A label-free method based on MALDI-TOF mass spectrometry for the absolute quantitation of troponin T in mouse cardiac tissue.
A label-free absolute quantitation method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been developed. This methodology was applied to mouse heart in order to quantify cardiac troponin T (cTnT), which is considered to be a sensitive marker of heart damage. The cTnT was extracted, isolated by reversed-phase high-performance liquid chromatography, digested, and analyzed by MALDI-TOF MS. The MS-based quantitation was performed using matrix-matched calibration curves (due to a matrix effect) of two synthetic peptides, one cTnT-specific peptide and one internal standard peptide, respectively. Recoveries at three spiking levels ranged from 87-96%, with relative standard deviations of below 10%. The method detection limit and the method quantitation limit, expressed as the amount of cTnT for the amount of total sarcomeric protein extract, were 0.03 mg g(-1) and 0.15 mg g(-1), respectively. This method appears to be accurate and generally suitable for improving absolute protein quantitation.
Bizzarri
Mariano
M
Department of Experimental Pathology, "La Sapienza" University, via Scarpa 16, 00161, Roma, Italy.
Cavaliere
Chiara
C
Foglia
Patrizia
P
Guarino
Chiara
C
Samperi
Roberto
R
Laganà
Aldo
A
D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D008297:Male; D051379:Mice; D009206:Myocardium / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D020107:Troponin T / Q000032:analysis*
D020107:Troponin T