Luxembourg University, LU
Introduction
Targeted proteomics analyses in biomarker evaluation studies are routinely performed on triple quadrupole mass spectrometers in selected reaction monitoring (SRM) mode. However, the low resolution of quadrupole mass filters has limited selectivity for the analysis of complex samples (such as bodily fluids), characterized by a high background interfering with the signal of analytes. Hybrid mass spectrometers with high resolution and accurate mass (HRAM) capabilities overcome this limitation, and opened new avenues in quantitative proteomics. Targeted analyses of clinical samples were performed using parallel reaction monitoring (PRM) on a quadrupole-orbitrap mass spectrometer to demonstrate the gain in selectivity, while identifying the fragments by accurate mass, thus increasing the confidence in the measurements.
Results
Targeted LC-MS/MS analyses were first performed on a pool of control plasma samples. The quadrupole-orbitrap instrument operated in parallel reaction monitoring mode to acquire full MS/MS spectra of predefined precursor ions corresponding to the peptides of interest. The signals of fragment ions were extracted post-acquisition to obtain a series of extracted chromatograms equivalent to the traces of multiple transitions in SRM analyses. The use of narrow mass window to extract ion chromatograms (typically 10 ppm) dramatically reduced background interferences and improved the selectivity of the assays. The trapping capability of the quadrupole-orbitrap instrument proved beneficial for the enrichment of precursor ions of peptides present in tiny amounts and thus improved the signal-to-noise ratio of the signals. The impact of the quadrupole isolation window width on the selectivity of measurements was also evaluated. The increased transmission efficiency of the second generation quadrupole-orbitrap instrument allowed the use of narrower windows (< 2 Th) and thus better selectivity. In addition, the experimental design of experiments was greatly simplified as only m/z values of selected precursor ions were required instead of all predefined transitions with SRM.
A total of 80 peptides corresponding to 40 proteins, including lung cancer candidate markers, were monitored in triplicated PRM analyses of 10 patient (at different stages of the disease) and 10 control samples. The analyses were replicated in SRM mode on triple quadrupole instrument. The PRM analyses showed better quantification performance in most cases, by allowing more confident measurements of the endogenous peptides present in the lowest abundance. This translated in more consistent quantitative data for the different peptides surrogate of the same protein and a clear discrimination between the control and patient samples, and even different disease stages.