Biomarker discovery and validation are the first steps in understanding disease and drug development. Validation is technologically challenged since it requires analyzing a large number of samples with high-throughput, but also requires high sensitivity, high resolution, large dynamic range and excellent selectivity. Targeted LC-MS based assays afford protein quantification with the reproducibility and throughput required in order to improve biomarker acceptance. MRM, using both low, high and ion mobility enabled high resolution mass spectrometry, are enabling technologies. Miniaturized LC systems offer improved mass-sensitivity but often lack throughput, robustness and reproducibility. Here, we compare the application of an integrated microfluidic device with nanoscale LC, using different MS platforms, for the quantification of marker peptides and proteins, considering speed, sensitivity and selectivity.
Important aspects when characterizing LC-MS systems for validation/translation experiments are retention time reproducibility, technical reproducibility of the monitored transitions, and consistent, quantitative measurement accuracy. MRM transitions were inspected ensuring that a minimum of two transitions per peptide were detected. Typical retention time reproducibility for the monitored peptides was shown to be acceptable at microfluidic and nanoscale LC system levels and equaled 0.02 min standard deviation. Ratio measurements experimentally determined for peptides present in the MS Qual/Quant mixture were in good agreement with the manufacturer specified values. For all peptides, the average variation from the expected values was on average 11.5%. The levels of various putative peptide biomarkers in tryptically digested human serum matrix were determined, for example, ITLYGR was present at 650 amol per 200 ng of plasma matrix using the microfluic LC system in combination with low resolution tandem mass spectrometry, and GYSIFSYATK present at 45 amol per 200 ng of matrix using the nanoscale LC system in combination with high resolution Q-Tof mass spectrometry, operating with and without ion mobility enhancements.
Comprehensive comparison of various LC-MS platforms for the quantitative analysis of potential peptide biomarkers, including ion mobility based MRM technology.