title: High-performance hybrid Orbitrap mass spectrometers for quantitative proteome analysis: Observations and implications.
abstract: We present basic workups and quantitative comparisons for two current generation Orbitrap mass spectrometers, the Q Exactive Plus and Orbitrap Fusion Tribrid, which are widely considered two of the highest performing instruments on the market. We assessed the performance of two quantitative methods on both instruments, namely label-free quantitation and stable isotope labeling using isobaric tags, for studying the heat shock response in Escherichia coli. We investigated the recently reported MS3 method on the Fusion instrument and the potential of MS3-based reporter ion isolation Synchronous Precursor Selection (SPS) and its impact on quantitative accuracy. We confirm that the label-free approach offers a more linear response with a wider dynamic range than MS/MS-based isobaric tag quantitation and that the MS3/SPS approach alleviates but does not eliminate dynamic range compression. We observed, however, that the choice of quantitative approach had little impact on the ability to statistically evaluate the E. coli heat shock response. We conclude that in the experimental conditions tested, MS/MS-based reporter ion quantitation provides reliable biological insight despite the issue of compressed dynamic range, an observation that significantly impacts the choice of instrument.
authors.affiliation: Protein Research Group, VILLUM Center for Bioanalytical Sciences and Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
authors.forename: James C
authors.initials: JC
authors.lastname: Williamson
authors.affiliation: Protein Research Group, VILLUM Center for Bioanalytical Sciences and Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
authors.forename: Alistair V G
authors.initials: AV
authors.lastname: Edwards
authors.affiliation: Protein Research Group, VILLUM Center for Bioanalytical Sciences and Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.|Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Thiago
authors.initials: T
authors.lastname: Verano-Braga
authors.affiliation: Protein Research Group, VILLUM Center for Bioanalytical Sciences and Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
authors.forename: Veit
authors.initials: V
authors.lastname: Schwämmle
authors.affiliation: Protein Research Group, VILLUM Center for Bioanalytical Sciences and Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
authors.forename: Frank
authors.initials: F
authors.lastname: Kjeldsen
authors.affiliation: Protein Research Group, VILLUM Center for Bioanalytical Sciences and Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
authors.forename: Ole N
authors.initials: ON
authors.lastname: Jensen
authors.affiliation: Protein Research Group, VILLUM Center for Bioanalytical Sciences and Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
authors.forename: Martin R
authors.initials: MR
authors.lastname: Larsen
mesh_terms: D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000032:analysis / Q000378:metabolism; D018869:Heat-Shock Response; D020543:Proteome / Q000032:analysis* / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D029968:Escherichia coli Proteins; D020543:Proteome
keywords: Instrument optimization; Mass spectrometry instrumentation; Orbitrap fusion; Q Exactive Plus; Quantitation; Technology