Translational and biomarker verification studies are challenged in that they not only require the analysis of large sample cohorts with high-throughput, but also demand high sensitivity, high resolution and selectivity over a large dynamic range. Targeted LC-MS/MS based assays afford analyte quantification with the reproducibility and throughput required in order to rapidly assess biomarker performance. Multiple Reaction Monitoring (MRM), using tandem quadrupole mass spectrometry, is an enabling technology that provides speed and selectivity, whilst miniaturized LC systems offer additional improved sensitivity. Here, the application of micro-fluidics coupled to a novel tandem quadrupole MS/MS system, using a multi-point internal calibration method for the quantitation of peptides and proteins is presented and considered for speed, sensitivity, accuracy/ bias and selectivity.
Initial benchmarking of the MS system, fitted with a novel ion guide that enables improved and selective ion transmission, alongside with detector advances, was conducted by contrasting the sensitivity and precision performance for six stable isotope labelled peptides spike into biological matrix (tryptic digested, undepleted human plasma) against four other MS systems, including two tandem quadrupole and two high-resolution time-of-flight instruments, all operated in MRM mode of acquisition. On average, S/N was found to be two-fold better compared to the next most sensitive instrument in MRM mode of acquisition. %CV performance values were found to be similar to the two other tandem quadrupole instruments and approximately two fold better than the time-of-flight instruments.
Performance metrics of the method will be demonstrated and the results contrasted with those obtained with conventional MRM quantitation approaches for the direct analysis, without applying any type of fractionation or enrichment, of three human plasma peptides, representing putative blood-based markers of cardiovascular disease that span over five orders of dynamic concentration range.