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

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.

[1]  B. Ueberheide,et al.  Detection and correction of interference in SRM analysis. , 2013, Methods.

[2]  Eric W. Deutsch,et al.  A repository of assays to quantify 10,000 human proteins by SWATH-MS , 2014, Scientific Data.

[3]  Jacob D. Jaffe,et al.  Quantitative Assessment of Chromatin Immunoprecipitation Grade Antibodies Directed against Histone Modifications Reveals Patterns of Co-occurring Marks on Histone Protein Molecules* , 2012, Molecular & Cellular Proteomics.

[4]  Adele Bourmaud,et al.  Technical considerations for large-scale parallel reaction monitoring analysis. , 2014, Journal of proteomics.

[5]  Susan E Abbatiello,et al.  Automated detection of inaccurate and imprecise transitions in peptide quantification by multiple reaction monitoring mass spectrometry. , 2010, Clinical chemistry.

[6]  Michael J. MacCoss,et al.  Platform-independent and Label-free Quantitation of Proteomic Data Using MS1 Extracted Ion Chromatograms in Skyline , 2012, Molecular & Cellular Proteomics.

[7]  Bruno Domon,et al.  Large-Scale Targeted Proteomics Using Internal Standard Triggered-Parallel Reaction Monitoring (IS-PRM)* , 2015, Molecular & Cellular Proteomics.

[8]  Andrew Keller,et al.  Automated Validation of Results and Removal of Fragment Ion Interferences in Targeted Analysis of Data-independent Acquisition Mass Spectrometry (MS) using SWATHProphet* , 2015, Molecular & Cellular Proteomics.

[9]  M. Mann,et al.  Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics* , 2002, Molecular & Cellular Proteomics.

[10]  John Chilton,et al.  Using iRT, a normalized retention time for more targeted measurement of peptides , 2012, Proteomics.

[11]  Loïc Dayon,et al.  Isobaric tagging-based selection and quantitation of cerebrospinal fluid tryptic peptides with reporter calibration curves. , 2010, Analytical chemistry.

[12]  D R Mani,et al.  Simplified and Efficient Quantification of Low-abundance Proteins at Very High Multiplex via Targeted Mass Spectrometry* , 2014, Molecular & Cellular Proteomics.

[13]  R. Aebersold,et al.  High Sensitivity Detection of Plasma Proteins by Multiple Reaction Monitoring of N-Glycosites*S , 2007, Molecular & Cellular Proteomics.

[14]  Brendan MacLean,et al.  Bioinformatics Applications Note Gene Expression Skyline: an Open Source Document Editor for Creating and Analyzing Targeted Proteomics Experiments , 2022 .

[15]  Ben C. Collins,et al.  A tool for the automated, targeted analysis of data-independent acquisition MS-data: OpenSWATH , 2014 .

[16]  Matthias Mann,et al.  Fifteen years of Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC). , 2014, Methods in molecular biology.

[17]  Hikaru Tsuchiya,et al.  The parallel reaction monitoring method contributes to a highly sensitive polyubiquitin chain quantification. , 2013, Biochemical and biophysical research communications.

[18]  Steven A. Carr,et al.  Building the Connectivity Map of epigenetics: chromatin profiling by quantitative targeted mass spectrometry. , 2015, Methods.

[19]  S. Carr,et al.  Quantitative analysis of peptides and proteins in biomedicine by targeted mass spectrometry , 2013, Nature Methods.

[20]  K. Parker,et al.  Multiplexed Protein Quantitation in Saccharomyces cerevisiae Using Amine-reactive Isobaric Tagging Reagents*S , 2004, Molecular & Cellular Proteomics.

[21]  S. Carr,et al.  Quantitative, Multiplexed Assays for Low Abundance Proteins in Plasma by Targeted Mass Spectrometry and Stable Isotope Dilution*S , 2007, Molecular & Cellular Proteomics.

[22]  R. Aebersold,et al.  mProphet: automated data processing and statistical validation for large-scale SRM experiments , 2011, Nature Methods.

[23]  Christopher M Rose,et al.  NeuCode Labels for Relative Protein Quantification * , 2014, Molecular & Cellular Proteomics.

[24]  Dylan J. Sorensen,et al.  Structural, Kinetic and Proteomic Characterization of Acetyl Phosphate-Dependent Bacterial Protein Acetylation , 2014, PloS one.

[25]  Jun Fan,et al.  A critical appraisal of techniques, software packages, and standards for quantitative proteomic analysis. , 2012, Omics : a journal of integrative biology.

[26]  Ludovic C. Gillet,et al.  Targeted Data Extraction of the MS/MS Spectra Generated by Data-independent Acquisition: A New Concept for Consistent and Accurate Proteome Analysis* , 2012, Molecular & Cellular Proteomics.

[27]  Michael S. Bereman,et al.  Implementation of Statistical Process Control for Proteomic Experiments Via LC MS/MS , 2014, Journal of The American Society for Mass Spectrometry.

[28]  R. Aebersold,et al.  Selected reaction monitoring for quantitative proteomics: a tutorial , 2008, Molecular systems biology.

[29]  Pei Wang,et al.  Demonstrating the feasibility of large-scale development of standardized assays to quantify human proteins , 2013, Nature Methods.

[30]  Brendan MacLean,et al.  Label-Free Quantitation of Protein Modifications by Pseudo Selected Reaction Monitoring with Internal Reference Peptides , 2012, Journal of proteome research.

[31]  Allan R Brasier,et al.  Multiplexed parallel reaction monitoring targeting histone modifications on the QExactive mass spectrometer. , 2014, Analytical chemistry.

[32]  D. S. Hage,et al.  System suitability in bioanalytical LC/MS/MS. , 2007, Journal of pharmaceutical and biomedical analysis.

[33]  Derek J. Bailey,et al.  Parallel Reaction Monitoring for High Resolution and High Mass Accuracy Quantitative, Targeted Proteomics* , 2012, Molecular & Cellular Proteomics.

[34]  Christoph H Borchers,et al.  Design, Implementation and Multisite Evaluation of a System Suitability Protocol for the Quantitative Assessment of Instrument Performance in Liquid Chromatography-Multiple Reaction Monitoring-MS (LC-MRM-MS)* , 2013, Molecular & Cellular Proteomics.

[35]  Brendan MacLean,et al.  Panorama: A Targeted Proteomics Knowledge Base , 2014, Journal of proteome research.

[36]  Marco Y. Hein,et al.  Accurate Proteome-wide Label-free Quantification by Delayed Normalization and Maximal Peptide Ratio Extraction, Termed MaxLFQ * , 2014, Molecular & Cellular Proteomics.

[37]  B. Domon,et al.  Targeted Proteomic Quantification on Quadrupole-Orbitrap Mass Spectrometer* , 2012, Molecular & Cellular Proteomics.

[38]  Ruedi Aebersold,et al.  Conserved Peptide Fragmentation as a Benchmarking Tool for Mass Spectrometers and a Discriminating Feature for Targeted Proteomics* , 2014, Molecular & Cellular Proteomics.

[39]  B. Domon,et al.  Detection and quantification of proteins in clinical samples using high resolution mass spectrometry. , 2015, Methods.

[40]  T. Nawy Microbiology: Microbial planet , 2013, Nature Methods.

[41]  Dylan J. Sorensen,et al.  Label-Free Quantitation and Mapping of the ErbB2 Tumor Receptor by Multiple Protease Digestion with Data-Dependent (MS1) and Data-Independent (MS2) Acquisitions , 2013, International journal of proteomics.

[42]  Ruedi Aebersold,et al.  Reproducible Quantification of Cancer-Associated Proteins in Body Fluids Using Targeted Proteomics , 2012, Science Translational Medicine.