Parallel Reaction Monitoring for High Resolution and High Mass Accuracy Quantitative, Targeted Proteomics

Abstract

Selected reaction monitoring on a triple quadrupole mass spectrometer is currently experiencing a renaissance within the proteomics community for its, as yet, unparalleled ability to characterize and quantify a set of proteins reproducibly, completely, and with high sensitivity. Given the immense benefit that high resolution and accurate mass instruments have brought to the discovery proteomics field, we wondered if highly accurate mass measurement capabilities could be leveraged to provide benefits in the targeted proteomics domain as well. Here, we propose a new targeted proteomics paradigm centered on the use of next generation, quadrupole-equipped high resolution and accurate mass instruments: parallel reaction monitoring (PRM). In PRM, the third quadrupole of a triple quadrupole is substituted with a high resolution and accurate mass mass analyzer to permit the parallel detection of all target product ions in one, concerted high resolution mass analysis. We detail the analytical performance of the PRM method, using a quadrupoleequipped bench-top Orbitrap MS, and draw a performance comparison to selected reaction monitoring in terms of run-to-run reproducibility, dynamic range, and measurement accuracy. In addition to requiring minimal upfront method development and facilitating automated data analysis, PRM yielded quantitative data over a wider dynamic range than selected reaction monitoring in the presence of a yeast background matrix because of PRM's high selectivity in the mass-to-charge domain. With achievable linearity over the quantifiable dynamic range found to be statistically equal between the two methods, our investigation suggests that PRM will be a promising new addition to the quantitative proteomics toolbox. Molecular & Cellular Proteomics 11: 10.1074/ mcp.O112.020131, 1475-1488, 2012.The most widespread protein sequencing technique is the shotgun method. Proteins are digested into peptides, chromatographically separated, and measured by mass spectrometers (1-10). Many types of mass spectrometers are used-quadrupole ion traps, quadrupole ion trap hybrids such as the QLT (quadrupole linear ion trap)-Orbitrap or QLT-FT-ICR, and quadrupole time-of-flight (Q-TOF) hybrids-but, the experiments, from the MS measurement onward, are basically identical: the masses of eluting cationic peptide precursors are measured in a MS scan, and the most abundant precursors are selected in series for successive tandem MS events (MS/MS). This process, called data-dependent acquisition, continues for the duration of a chromatographic separation, and constant MS operation in this manner can generate hundreds of thousands of spectra in days. These spectra are then mapped to peptide or protein sequence databases using highly-evolved database search algorithms (11-13). Successful results can be obtained within just a few days and are nothing short of spectacular: tens of thousands of unique peptide spectral matches mapping to several thousand unique protein isoforms have become the norm. Although this approach certainly ...

High mass accuracy assay for trimethylamine N-oxide using stable-isotope dilution with liquid chromatography coupled to orthogonal acceleration time of flight mass spectrometry with multiple reaction monitoring

“…ToF-MRM assays have the inherent capability to be multiplexed and thus have incorporated into a single analysis these related analytes and their respective isotopically labelled standards for parallel quantitative measurement. High level selectivity that may be required to delineate some species in multiplex assays, those displaying extremely similar retention time/mass-to-charge profiles, is benefited by employment of the high resolution and high mass accuracy that is afforded by new generation Q-ToF or Q-Orbitrap instruments [30][31][32].…”

Analysis of Major Histocompatibility Complex (MHC) Immunopeptidomes Using Mass Spectrometry*

“…Stateof-the-art targeted methods are mainly performed in S/MRM and PRM mode on a low-resolution triple quadrupole and a high-resolution quadrupole Orbitrap mass spectrometer, respectively (Fig. 2, middle panel) (84,86). Such MS-based targeted platforms require a priori knowledge of the molecular targets.…”

Detection of Allergen Markers in Food: Analytical Methods

“…Variants of MRM assays can also exist for ion trap instruments (pseudo-selected reaction monitoring, pSRM) or quadrupole-Orbitrap hybrid instruments (parallel reaction monitoring, PRM). PRM is a targeted proteomics strategy where all products of a precursor peptide are simultaneously monitored under conditions that offer high resolution and ff ff high mass accuracy [155]. Preliminary reports suggest that PRM analyses exhibit dynamic range and performance characteristics that rival those of MRM analyses performed on triple quadrupole instruments [155,156].…”