Development and analytical comparison of microflow and nanoflow liquid chromatography/mass spectrometry procedures for quantification of cardiac troponin T in mouse hearts

Abstract

Three procedures for the quantification of cardiac troponin T (cTnT) based on liquid chromatography/mass spectrometry (LC/MS) were developed, validated and compared. The procedures were applied to estimate the cTnT content in the hearts of wild type mice C57BL/6J (WT) and double knock-outs for apolipoprotein E and receptor for LDL (AL KO).Three variants of the procedure proposed include microflow, direct injection nanoflow and preconcentration nanoflow LC/MS. Troponin T tryptic peptide YEINVLR and its analog (internal standard) were monitored in a multiple reaction monitoring mode using triple quadrupole mass detector with electrospray (ESI) ion sources.The preconcentration nanoflow LC/MS method offered the best sensitivity with a lower limit of quantification (LLOQ) of 0.25fgµL−1 and a minimal matrix effect. The LLOQ value was 8 times better, compared with that in direct injection nanoflow LC/MS and 200 times better than in microflow LC/MS. The accuracy or precision for all three methods were not different. Separation time in the direct injection nanoflow (8min) was equivalent to the microflow method (6min). The cTnT contents in the mice hearts measured by the methods developed by the present authors were not different between the WT and AL KO.We conclude that nanoflow LC/MS based quantitative proteomics offers fundamentally better sensitivities while maintaining analytical quality and separation times equivalent to microflow procedures.