Targeted Proteomic Quantification on Quadrupole-Orbitrap Mass Spectrometer

There is an immediate need for improved methods to systematically and precisely quantify large sets of peptides in complex biological samples. To date protein quantification in biological samples has been routinely performed on triple quadrupole instruments operated in selected reaction monitoring mode (SRM), and two major challenges remain. Firstly, the number of peptides to be included in one survey experiment needs to be increased to routinely reach several hundreds, and secondly, the degree of selectivity should be improved so as to reliably discriminate the targeted analytes from background interferences. High resolution and accurate mass (HR/AM) analysis on the recently developed Q-Exactive mass spectrometer can potentially address these issues. This instrument presents a unique configuration: it is constituted of an orbitrap mass analyzer equipped with a quadrupole mass filter as the front-end for precursor ion mass selection. This configuration enables new quantitative methods based on HR/AM measurements, including targeted analysis in MS mode (single ion monitoring) and in MS/MS mode (parallel reaction monitoring). The ability of the quadrupole to select a restricted m/z range allows one to overcome the dynamic range limitations associated with trapping devices, and the MS/MS mode provides an additional stage of selectivity. When applied to targeted protein quantification in urine samples and benchmarked with the reference SRM technique, the quadrupole-orbitrap instrument exhibits similar or better performance in terms of selectivity, dynamic range, and sensitivity. This high performance is further enhanced by leveraging the multiplexing capability of the instrument to design novel acquisition methods and apply them to large targeted proteomic studies for the first time, as demonstrated on 770 tryptic yeast peptides analyzed in one 60-min experiment. The increased quality of quadrupole-orbitrap data has the potential to improve existing protein quantification methods in complex samples and address the pressing demand of systems biology or biomarker evaluation studies. There is an immediate need for improved methods to systematically and precisely quantify large sets of peptides in complex biological samples. To date protein quantification in biological samples has been routinely performed on triple quadrupole instruments operated in selected reaction monitoring mode (SRM), and two major challenges remain. Firstly, the number of peptides to be included in one survey experiment needs to be increased to routinely reach several hundreds, and secondly, the degree of selectivity should be improved so as to reliably discriminate the targeted analytes from background interferences. High resolution and accurate mass (HR/AM) analysis on the recently developed Q-Exactive mass spectrometer can potentially address these issues. This instrument presents a unique configuration: it is constituted of an orbitrap mass analyzer equipped with a quadrupole mass filter as the front-end for precursor ion mass selection. This configuration enables new quantitative methods based on HR/AM measurements, including targeted analysis in MS mode (single ion monitoring) and in MS/MS mode (parallel reaction monitoring). The ability of the quadrupole to select a restricted m/z range allows one to overcome the dynamic range limitations associated with trapping devices, and the MS/MS mode provides an additional stage of selectivity. When applied to targeted protein quantification in urine samples and benchmarked with the reference SRM technique, the quadrupole-orbitrap instrument exhibits similar or better performance in terms of selectivity, dynamic range, and sensitivity. This high performance is further enhanced by leveraging the multiplexing capability of the instrument to design novel acquisition methods and apply them to large targeted proteomic studies for the first time, as demonstrated on 770 tryptic yeast peptides analyzed in one 60-min experiment. The increased quality of quadrupole-orbitrap data has the potential to improve existing protein quantification methods in complex samples and address the pressing demand of systems biology or biomarker evaluation studies. Shotgun proteomics has emerged over the past decade as the most effective method for the qualitative study of complex proteomes (i.e., the identification of the protein content), as illustrated by a wealth of publications (1Aebersold R. Mann M. Mass spectrometry-based proteomics.Nature. 2003; 422: 198-207Crossref PubMed Scopus (5585) Google Scholar, 2Domon B. Aebersold R. Mass spectrometry and protein analysis.Science. 2006; 312: 212-217Crossref PubMed Scopus (1610) Google Scholar). In this approach, after enzymatic digestion of the proteins, the generated peptides are analyzed by means of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) 1The abbreviations used are:SRMselected reaction monitoringSIMsingle ion monitoringMSmass spectrometryMS/MStandem mass spectrometryLCliquid chromatographyHCDhigher energy collisional dissociationHR/AMhigh resolution/accurate massLOQlimit of quantificationAGCautomatic gain controlPRMparallel reaction monitoringAUCarea under the curveCVcoefficient of variation. 1The abbreviations used are:SRMselected reaction monitoringSIMsingle ion monitoringMSmass spectrometryMS/MStandem mass spectrometryLCliquid chromatographyHCDhigher energy collisional dissociationHR/AMhigh resolution/accurate massLOQlimit of quantificationAGCautomatic gain controlPRMparallel reaction monitoringAUCarea under the curveCVcoefficient of variation.in a data dependent mode. However, the complexity of the digested proteomes under investigation and the wide range of protein abundances limit the reproducibility and the sensitivity of this stochastic approach (3Aebersold R. A stress test for mass spectrometry-based proteomics.Nat. Methods. 2009; 6: 411-412Crossref PubMed Scopus (47) Google Scholar), which is critical if one aims the quantification of the MS emerged for the quantitative study of complex the targeted proteomics B. Aebersold R. and a quantitative proteomics PubMed Scopus Google Scholar). In this approach, of analytes targeted peptides used as for the of are in m/z and which the most with When applied to complex biological as urine or proteomics high performance instruments of a wide dynamic range of with high sensitivity in to peptides in the range and selectivity to with background The and PubMed Scopus Google Scholar). reaction monitoring on triple quadrupole quadrupole mass spectrometry for analysis and PubMed Scopus Google or triple ion mass ion a ion mass Mass 2003; PubMed Scopus Google has emerged as a means to B. reaction monitoring applied to Mass PubMed Scopus Google Scholar). applied in the MS analysis of in and by in mass spectrometry-based Mass Scopus Google Scholar, R. spectrometry in Mass 2003; PubMed Scopus Google Scholar), SRM has emerged as the reference quantitative for in biological samples. 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