Increased Selectivity, Analytical Precision, and Throughput in Targeted Proteomics

Proteomics is gradually complementing large shotgun qualitative studies with hypothesis-driven quantitative experiments. Targeted analyses performed on triple quadrupole instruments in selected reaction monitoring mode are characterized by a high degree of selectivity and low limit of detection; however, the concurrent analysis of multiple analytes occurs at the expense of sensitivity because of reduced dwell time and/or selectivity due to limitation to a few transitions. A new data acquisition paradigm is presented in which selected reaction monitoring is performed in two ways to simultaneously quantify and confirm the identity of the targeted peptides. A first set of primary transitions is continuously monitored during a predetermined elution time window to precisely quantify each peptide. In addition, a set of six to eight transitions is acquired in a data-dependent event, triggered when all the primary transitions exceed a preset threshold. These additional transitions are used to generate composite tandem mass spectra to formally confirm the identity of the targeted peptides. This technique was applied to analyze the tryptic digest of a yeast lysate to demonstrate the performance of the technique. We showed a limit of detection down to tens of attomoles injected and a throughput exceeding 6000 transitions in one 60-min experiment. The technique was integrated into a linear work flow, including experimental design, data acquisition, and data evaluation, enabling large scale proteomic studies. Proteomics is gradually complementing large shotgun qualitative studies with hypothesis-driven quantitative experiments. Targeted analyses performed on triple quadrupole instruments in selected reaction monitoring mode are characterized by a high degree of selectivity and low limit of detection; however, the concurrent analysis of multiple analytes occurs at the expense of sensitivity because of reduced dwell time and/or selectivity due to limitation to a few transitions. A new data acquisition paradigm is presented in which selected reaction monitoring is performed in two ways to simultaneously quantify and confirm the identity of the targeted peptides. A first set of primary transitions is continuously monitored during a predetermined elution time window to precisely quantify each peptide. In addition, a set of six to eight transitions is acquired in a data-dependent event, triggered when all the primary transitions exceed a preset threshold. These additional transitions are used to generate composite tandem mass spectra to formally confirm the identity of the targeted peptides. This technique was applied to analyze the tryptic digest of a yeast lysate to demonstrate the performance of the technique. We showed a limit of detection down to tens of attomoles injected and a throughput exceeding 6000 transitions in one 60-min experiment. The technique was integrated into a linear work flow, including experimental design, data acquisition, and data evaluation, enabling large scale proteomic studies. Proteomics is gradually complementing qualitative studies focused on protein identification relying on shotgun strategies (1.Aebersold R. Mann M. Mass spectrometry-based proteomics.Nature. 2003; 422: 198-207Crossref PubMed Scopus (5585) Google Scholar, 2.Lin D. Tabb D.L. Yates 3rd, J.R. Large-scale protein identification using mass spectrometry.Biochim. Biophys. Acta. 2003; 1646: 1-10Crossref PubMed Scopus (191) Google Scholar) with large scale quantitative experiments. This change was prompted by the growing demand for qualification and verification of putative protein biomarkers through analysis of larger cohorts of clinical samples on one hand and the need for consistent quantitative data sets to facilitate modeling in systems biology studies on the other. In either case, the number of proteins under target is quite large (tens to hundreds), and traditional immunoassay approaches are not suited for use because of the cost, time, and difficulty of developing multiplexed assays. In this context, the selected reaction monitoring (SRM) 1The abbreviations used are:SRMselected reaction monitoringiSRMintelligent selected reaction monitoringt-SRMtime-based SRMCVcoefficient of variation. 1The abbreviations used are:SRMselected reaction monitoringiSRMintelligent selected reaction monitoringt-SRMtime-based SRMCVcoefficient of variation. technique performed on a triple quadrupole mass spectrometer is increasingly applied to quantitative proteomics because of its sensitivity, selectivity, and wide dynamic range (3.Domon B. Aebersold R. Mass spectrometry and protein analysis.Science. 2006; 312: 212-217Crossref PubMed Scopus (1610) Google Scholar, 4.Anderson L. Hunter C.L. Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins.Mol. Cell. Proteomics. 2006; 5: 573-588Abstract Full Text Full Text PDF PubMed Scopus (1078) Google Scholar, 5.Keshishian H. Addona T. Burgess M. Kuhn E. Carr S.A. Quantitative, multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution.Mol. Cell. Proteomics. 2007; 6: 2212-2229Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar, 6.Kuzyk M.A. Smith D. Yang J. Cross T.J. Jackson A.M. Hardie D.B. Anderson N.L. Borchers C.H. MRM-based, multiplexed, absolute quantitation of 45 proteins in human plasma.Mol. Cell. Proteomics. 2009; 8: 1860-1877Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). Mass spectrometry assays can be developed very rapidly via the use of commodity synthetic reference peptides libraries (7.Picotti P. Rinner O. Stallmach R. Dautel F. Farrah T. Domon B. Wenschuh H. Aebersold R. High-throughput generation of selected reaction-monitoring assays for proteins and proteomes.Nat. Methods. 2010; 7: 43-46Crossref PubMed Scopus (399) Google Scholar), and large resources of peptide MS/MS data (MRMAtlas) (8.Picotti P. Lam H. Campbell D. Deutsch E.W. Mirzaei H. Ranish J. Domon B. Aebersold R. A database of mass spectrometric assays for the yeast proteome.Nat. Methods. 2008; 5: 913-914Crossref PubMed Scopus (182) Google Scholar) are available to design the initial assays. However, developing a robust and precise SRM-based multiple assay remains demanding for proteomics experiments. selected reaction monitoring intelligent selected reaction monitoring time-based SRM coefficient of variation. selected reaction monitoring intelligent selected reaction monitoring time-based SRM coefficient of variation. One of the main challenges is that most proteomics samples are highly complex, and several interfering signals are detected within a given time window that require systematic verification of the target peptide identity first to ensure its accurate quantification. Using isotopically labeled counterparts of the targeted analytes is a common way to confirm the target peptide identity (9.Gerber S.A. Rush J. Stemman O. Kirschner M.W. Gygi S.P. Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 6940-6945Crossref PubMed Scopus (1542) Google Scholar, 10.Anderson N.L. Anderson N.G. Haines L.R. Hardie D.B. Olafson R.W. Pearson T.W. Mass spectrometric quantitation of peptides and proteins using stable isotope standards and capture by anti-peptide antibodies (SISCAPA).J. Proteome Res. 2004; 3: 235-244Crossref PubMed Scopus (694) Google Scholar). As this may not always be practical for large scale quantitative proteomics studies, an alternative way to verify the peptide identity is to use SRM-initiated full MS/MS scans (11.Unwin R.D. Griffiths J.R. Whetton A.D. A sensitive mass spectrometric method for hypothesis-driven detection of peptide post-translational modifications: multiple reaction monitoring-initiated detection and sequencing (MIDAS).Nat. Protoc. 2009; 4: 870-887Crossref PubMed Scopus (82) Google Scholar). However, the disadvantage of this method is a lower sensitivity and selectivity compared with SRM as it uses a broader mass selection window, which results in MS/MS spectra often containing signals from multiple components co-eluting in the case of biological samples with a complex background. Furthermore, SRM-initiated MS/MS scans require much longer duty cycle times that will disrupt the predefined SRM sequence of events in the case of complex multiplexed assays even when performed on a triple quadrupole instrument equipped with a linear ion trap. Recently, instead of using MS/MS spectra, a composite MS/MS spectrum that is generated by measuring multiple fragments ions (8–10 ions) from one specific peptide has been proven to provide sufficient information for peptide identification. 2Prakash, A., Kiyonami, R., Schoen, A., Nguyen, H. Q., Peterman, S. M., Huhmer, A., Lopez, M. F., Domon, B., and Aebersold, R. (2009) Integrated workflow to design methods and analyze data in large scale SRM experiments, Poster TH695 presented at the 57th American Society for Mass Spectrometry annual conference, Philadelphia (May 31–June 4, 2009). 2Prakash, A., Kiyonami, R., Schoen, A., Nguyen, H. Q., Peterman, S. M., Huhmer, A., Lopez, M. F., Domon, B., and Aebersold, R. (2009) Integrated workflow to design methods and analyze data in large scale SRM experiments, Poster TH695 presented at the 57th American Society for Mass Spectrometry annual conference, Philadelphia (May 31–June 4, 2009). The peptide is verified both by the overlay of the chromatographic elution profiles of the fragment ions and by matching the composite MS/MS spectrum that comprises multiple SRM transitions to the MS/MS spectral library entry (12.Prakash A. Tomazela D.M. Frewen B. Maclean B. Merrihew G. Peterman S. Maccoss M.J. Expediting the development of targeted SRM assays: using data from shotgun proteomics to automate method development.J. Proteome Res. 2009; 8: 2733-2739Crossref PubMed Scopus (120) Google Scholar, 13.Sherwood C.A. Eastham A. Lee L.W. Risler J. Vitek O. Martin D.B. Correlation between y-type ions observed in ion trap and triple quadrupole mass spectrometers.J. Proteome Res. 2009; 8: 4243-4251Crossref PubMed Scopus (46) Google Scholar). Because it is based on SRM acquisition, this method provides a rapid, sensitive, and selective way to perform peptide verification, which is desirable for large scale screening experiments. The drawback of this approach is that only a limited number of compounds can be in one because the instrument is continuously monitoring transitions for each peptide the peptide is detected from the in the of a of the instrument this and the that the instrument only a of SRM transitions for each peptide for the quantification and in confirm the identity using the full set of fragment which are acquired in a data-dependent provide this instrument developed an instrument intelligent selected reaction monitoring that can use the of a of SRM transitions to quantify and the full for of target the qualitative and quantitative analysis of to peptides in a experiment. the of and the work to and demonstrate the throughput the development of SRM assays and its to perform large scale a number of A yeast cell lysate was in containing at a of The proteins reduced with for at and with for 45 at in the with to a of and sequencing was to a of and for at by and with The peptide samples on a to and in to The yeast digest was used through all in this and was as P. B. Domon B. Aebersold R. Full dynamic range analysis of S. by targeted 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). A from was performed on a using a at and to in at a of The was generated by containing and containing One of each was performed on a triple quadrupole mass spectrometer in the a and The selectivity for both and set to The of was set at of The was using the of ion the selected reaction monitoring using the instrument of the two primary and an additional six fragment ions used for each targeted peptide. The instrument acquisition including elution time window, and for the data-dependent SRM generated by using first on the chromatographic A of isotopically labeled peptides and with the isotopically labeled was on the monitored all for peptides. Using the elution time for each peptide. In addition, for each its using the presented in R. for of times of tryptic peptides in its to protein peptide by Cell. Proteomics. 2004; 3: Full Text Full Text PDF PubMed Scopus Google Scholar). a linear was generated between the observed time and the for and the of was using the Using this linear for peptide first its using the and using the linear its elution using the elution time, a window for the transitions of this peptide. The generated method with the acquisition was to the instrument method instrument used for the cycle time, time, and dynamic set for the data-dependent SRM The cycle time of was used as The times used for the data-dependent SRM scans was with of threshold. The dynamic was set to the SRM events only when the of all primary SRM transitions for The dynamic time was performed based on the resources of peptide MS/MS data from each targeted the two most fragment ions selected as primary and an additional six fragments selected as A of transitions primary and used for eight peptide The isotopically labeled peptides into the yeast digest at and The analyses performed by of The can be in A of transitions primary and used for large scale peptides from The can be in A of transitions primary and for used for of and peptides in the cycle of The can be in by The targeted peptides verified by both chromatographic profiles of multiple fragment ions and the between the composite MS/MS spectrum that comprises multiple SRM transitions with the MS/MS spectral library The MS/MS spectral library was by in fragment ion data from the database of the targeted SRM data generated from a triple quadrupole mass spectrometer with MS/MS data of a peptide in a spectrum a spectrum library method was used (12.Prakash A. Tomazela D.M. Frewen B. Maclean B. Merrihew G. Peterman S. Maccoss M.J. Expediting the development of targeted SRM assays: using data from shotgun proteomics to automate method development.J. Proteome Res. 2009; 8: 2733-2739Crossref PubMed Scopus (120) Google Scholar). The for all SRM transitions and that to the ion in of the ions from the MS/MS library spectrum in of A A of J. Scopus Google Scholar) was as a of between the SRM data and the MS/MS spectrum in the library for the target is the coefficient range of are is the of the ion in the library spectrum and is the of the ion in the from the SRM and is the number of ions under provide an of the the coefficient was compared with library spectra and The was used to a of spectral using The a between the SRM data and spectral The peptide that a was to be by the library We used a of to only spectra that lower as using the library This was based on between large sets of composite spectra and spectra compared (12.Prakash A. Tomazela D.M. Frewen B. Maclean B. Merrihew G. Peterman S. Maccoss M.J. Expediting the development of targeted SRM assays: using data from shotgun proteomics to automate method development.J. Proteome Res. 2009; 8: 2733-2739Crossref PubMed Scopus (120) Google Scholar). each targeted peptide that the verification the the integrated of all primary ions for quantification and of for the experiments. The systematic and targeted analysis of proteomics samples has to two main sufficient selectivity has to be to quantify the of in complex biological which are characterized by through the monitoring of multiple transitions. the number of peptides to be in a given time window has to the cycle time for quantification. to be the dwell time of each that may need to be for low abundance ions and the number of transitions for each A between selectivity and sensitivity is a larger set of exceeding a is under The work the of selected reaction monitoring to simultaneously quantify and confirm the identity of the targeted peptides in two The of the acquisition method is in A few two to the most fragments in the MS/MS spectra of targeted to as primary are monitored continuously during a elution time window of each an is by the that can into the of the all the primary transitions all a is to additional transitions to as transitions that are used to generate a composite MS/MS spectrum to confirm that the signals observed for the primary transitions are to the targeted peptide as to interfering In the acquisition of transitions is performed when the is its a of to a few of is set the of the data-dependent This design of the dynamic each SRM acquisition to be triggered only for each which sufficient information to confirm the identity of the the quantification with the primary SRM The design of an SRM several including the selection of the peptides to the proteins that are targeted in the These tryptic peptides need to be with the proteins of In addition, need to specific detection in the mass as and an practical peptides containing that are each selected specific including time, ion fragment ion and are to an SRM a practical of MS/MS data facilitate the design of the method and the of SRM experiments, the spectra acquired on an instrument with compared with a triple is a of information available in proteomics MS/MS for E.W. H. N.L. B. Lee H. R. Aebersold R. plasma 5: PubMed Scopus Google Scholar) Proteome R. for and protein identification Proteome Res. 2004; 3: PubMed Scopus (576) Google Scholar). containing SRM transitions been (MRMAtlas) (8.Picotti P. Lam H. Campbell D. Deutsch E.W. Mirzaei H. Ranish J. Domon B. Aebersold R. A database of mass spectrometric assays for the yeast proteome.Nat. Methods. 2008; 5: 913-914Crossref PubMed Scopus (182) Google Scholar) that a primary to design experiments. information is to an the selection of primary and fragment ions in the of of peptides in an for the using to design the assay based on the data of a spectral library In this using a yeast cell lysate to demonstrate of the fragment ion information of each targeted peptide based on the database of by using the the in the information of fragment ion from the database to a MS/MS spectral the information of this spectral library was used to the primary and ions of each experiment. 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R.W. A. analysis of protein in 2003; PubMed Scopus Google to protein range in yeast using of two primary by S. R.W. A. analysis of protein in 2003; PubMed Scopus Google of two primary in a new These results compared with a performed on a of peptides that using the time-based SRM method in which all eight transitions monitored during the time window for peptide verification and quantification. 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