Multiplexed, Scheduled, High-Resolution Parallel Reaction Monitoring on a Full Scan QqTOF Instrument with Integrated Data-Dependent and Targeted Mass Spectrometric Workflows

Abstract

Recent advances in commercial mass spectrometers with higher resolving power and faster scanning capabilities have expanded their functionality beyond traditional data-dependent acquisition (DDA) to targeted proteomics with higher precision and multiplexing. Using an orthogonal quadrupole time-of flight (QqTOF) LC-MS system, we investigated the feasibility of implementing large-scale targeted quantitative assays using scheduled, high resolution multiple reaction monitoring (sMRM-HR), also referred to as parallel reaction monitoring (sPRM). We assessed the selectivity and reproducibility of PRM, also referred to as parallel reaction monitoring, by measuring standard peptide concentration curves and system suitability assays. By evaluating up to 500 peptides in a single assay, the robustness and accuracy of PRM assays were compared to traditional SRM workflows on triple quadrupole instruments. The high resolution and high mass accuracy of the full scan MS/MS spectra resulted in sufficient selectivity to monitor 6–10 MS/MS fragment ions per target precursor, providing flexibility in postacquisition assay refinement and optimization. The general applicability of the sPRM workflow was assessed in complex biological samples by first targeting 532 peptide precursor ions in a yeast lysate, and then 466 peptide precursors from a previously generated candidate list of differentially expressed proteins in whole cell lysates from E. coli. Lastly, we found that sPRM assays could be rapidly and efficiently developed in Skyline from DDA libraries when acquired on the same QqTOF platform, greatly facilitating their successful implementation. These results establish a robust sPRM workflow on a QqTOF platform to rapidly transition from discovery analysis to highly multiplexed, targeted peptide quantitation.

High-performance targeted mass spectrometry with precision data-independent acquisition reveals site-specific glycosylation macroheterogeneity

“…Alternatives to SRM for targeted proteomics using hybrid Q-TOF mass spectrometers include pseudo-MRM [29] and high resolution multiple reaction monitoring (MRM HR ) [30,31], with data typically analysed and quantified with MultiQuant™ or Skyline software. MRM HR or pseudo-MRM scan a set of predefined precursors and fragment them, allowing monitoring of multiple MS/MS fragment ions.…”

Oncogenic KRAS and BRAF Drive Metabolic Reprogramming in Colorectal Cancer

“…Multiplexed protein quantitation by multiple reaction monitoring (MRM) or parallel reaction monitoring (PRM) are powerful tools for systems characterization (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39). These multiplexed assays can interrogate coordinated expression of proteins in functional protein networks, such ␤-catenin signaling (35), nuclear factor-B signaling (36), protein expression changes because of EGFR signaling (37,38), and phosphotyrosine quantitation in EGFR signaling (39).…”

Prospective applications of ultrahigh resolution proteomics in clinical mass spectrometry

“…While accurate quantification of a set of proteins in body fluids is possible within each of these three concentration ranges, provided that suitable sample preparation strategies have been applied, a simultaneous procedure for the full concentration range is not possible. Coincidently, all high-end MS systems have a dynamic measurement range of 3-4 orders of magnitude, which matches with the rough separation of three concentration ranges of body fluids in 'high,' 'medium,' and 'low' [6]. The first three to four orders of magnitude (high concentration) can be covered without extensive up-front purification.…”

“…>100,000) were used by a small number of dedicated groups to characterize for example proteins and carbohydrates [2][3][4]. Meanwhile hybrid-type quadrupole time-of-flight (qTOF) mass spectrometers have evolved to currently available resolving powers up to 50,000 [5,6]. Both these high-and ultrahigh resolution mass spectrometers are referred to as HRAM platforms that have matured into a widely applied high-quality and large-scale procedure for structural analysis of almost any type of biomolecule.…”