Abstract
There is an immediate need for improved methods to systematically and precisely quantify large sets of peptides in complex biological samples. To date protein quantification in biological samples has been routinely performed on triple quadrupole instruments operated in selected reaction monitoring mode (SRM), and two major challenges remain. Firstly, the number of peptides to be included in one survey experiment needs to be increased to routinely reach several hundreds, and secondly, the degree of selectivity should be improved so as to reliably discriminate the targeted analytes from background interferences. High resolution and accurate mass (HR/AM) analysis on the recently developed Q-Exactive mass spectrometer can potentially address these issues. This instrument presents a unique configuration: it is constituted of an orbitrap mass analyzer equipped with a quadrupole mass filter as the front-end for precursor ion mass selection. This configuration enables new quantitative methods based on HR/AM measurements, including targeted analysis in MS mode (single ion monitoring) and in MS/MS mode (parallel reaction monitoring). The ability of the quadrupole to select a restricted m/z range allows one to overcome the dynamic range limitations associated with trapping devices, and the MS/MS mode provides an additional stage of selectivity. When applied to targeted protein quantification in urine samples and benchmarked with the reference SRM technique, the quadrupole-orbitrap instrument exhibits similar or better performance in terms of selectivity, dynamic range, and sensitivity. This high performance is further enhanced by leveraging the multiplexing capability of the instrument to design novel acquisition methods and apply them to large targeted proteomic studies for the first time, as demonstrated on 770 tryptic yeast peptides analyzed in one 60-min experiment. The increased quality of quadrupole-orbitrap data has the potential to improve existing protein quantification methods in complex samples and address the pressing demand of systems biology or biomarker evaluation studies.
Highlights
From the ‡Luxembourg Clinical Proteomics Center (LCP), CRPSante, L-1445 Strassen, Luxembourg; §Thermo Fisher Scientific, 28199 Bremen, Germany
For selected reaction monitoring mode (SRM) analysis on a triple quadrupole instrument, collecting all information required for method development can be a tedious process
For quadrupole-orbitrap analysis, two main modes of operation, which are discussed extensively below, were designed for targeted quantification: the single ion monitoring (SIM) mode and the parallel reaction monitoring4 (PRM) mode, relying on single-stage and tandem mass analysis, respectively. Both operation modes require a minimal set of instrument control parameters limited to the precursor ion m/z value and the predicted chromatographic retention time window
Summary
From the ‡Luxembourg Clinical Proteomics Center (LCP), CRPSante , L-1445 Strassen, Luxembourg; §Thermo Fisher Scientific, 28199 Bremen, Germany. High resolution/accurate mass (HR/AM) analysis represents a promising alternative approach that might more efficiently distinguish the compounds of interest from interferences in targeted proteomics Such analyses can be conducted on orbitrap-based mass spectrometers because of their high sensitivity and high mass accuracy capabilities [21]. The resulting data, acquired in the so-called single ion monitoring (SIM) mode, fully benefit from the trapping capability while keeping a high acquisition rate as a result of the fast switching time between targeted precursor ions of the quadrupole This mode of data acquisition is possible with a configuration combining a linear ion trap with the orbitrap (as in the LTQ-Orbitrap mass spectrometer), its effectiveness is far more limited in this case. The simultaneous monitoring of multiple MS/MS fragmentation channels, called parallel reaction monitoring (PRM), is promising for quantifying large sets of peptides with increased selectivity
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