title: 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.
authors.affiliation: Buck Institute for Research on Aging , 8001 Redwood Boulevard, Novato, California 94945, United States.
authors.forename: Birgit
authors.initials: B
authors.lastname: Schilling
authors.affiliation: Department of Genome Sciences, University of Washington School of Medicine , Foege Building S113, 3720 15th Avenue NE, Seattle, Washington 98195, United States.
authors.forename: Brendan
authors.initials: B
authors.lastname: MacLean
authors.affiliation: Departments of Medicine and Anesthesiology, Washington University School of Medicine , 660 South Euclid Avenue, St. Louis, Missouri 63110, United States.
authors.forename: Jason M
authors.initials: JM
authors.lastname: Held
authors.affiliation: Buck Institute for Research on Aging , 8001 Redwood Boulevard, Novato, California 94945, United States.
authors.forename: Alexandria K
authors.initials: AK
authors.lastname: Sahu
authors.affiliation: Buck Institute for Research on Aging , 8001 Redwood Boulevard, Novato, California 94945, United States.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Rardin
authors.affiliation: Buck Institute for Research on Aging , 8001 Redwood Boulevard, Novato, California 94945, United States.
authors.forename: Dylan J
authors.initials: DJ
authors.lastname: Sorensen
authors.affiliation: Buck Institute for Research on Aging , 8001 Redwood Boulevard, Novato, California 94945, United States.
authors.forename: Theodore
authors.initials: T
authors.lastname: Peters
authors.affiliation: Department of Microbiology and Immunology, Stritch School of Medicine, Health Sciences Division, Loyola University Chicago , 2160 South First Avenue, Maywood, Illinois 60153, United States.
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Wolfe
authors.affiliation: SCIEX, 1201 Radio Road, Redwood City, California 94065, United States.
authors.forename: Christie L
authors.initials: CL
authors.lastname: Hunter
authors.affiliation: Department of Genome Sciences, University of Washington School of Medicine , Foege Building S113, 3720 15th Avenue NE, Seattle, Washington 98195, United States.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: MacCoss
authors.affiliation: Buck Institute for Research on Aging , 8001 Redwood Boulevard, Novato, California 94945, United States.|Department of Pharmaceutical Chemistry, University of California , San Francisco, California 94143, United States.
authors.forename: Bradford W
authors.initials: BW
authors.lastname: Gibson
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000166:cytology; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000166:cytology; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000032:analysis*; D012441:Saccharomyces cerevisiae / Q000166:cytology; D012984:Software; D013997:Time Factors
chemical_list: D010455:Peptides