title: Mass measurement accuracy of the Orbitrap in intact proteome analysis.
abstract: The mass measurement accuracy (MMA) of Orbitrap mass spectrometers is 1-5 ppm according to the manufacturer's specification; yet, up to 50 ppm has been used as mass tolerance to interpret Orbitrap data in the literature. A systematic evaluation of MMA is thus necessary to find the optimal mass tolerance to be used.
  
  
  
  Reversed-phase liquid chromatography/tandem mass spectrometry (RPLC/MS/MS) analyses of the intact E. coli proteome were carried out on a Q Exactive Orbitrap mass spectrometer coupled to a Dionex UltiMate 3000 RSLCnano system. The analysis included three technical replicates each day and was repeated for six continuous days right after a mass calibration. The obtained raw datasets were searched using ProteinGoggle 2.0 under four different mass tolerances of 5, 10, 15, and 20 ppm.
  
  
  
  With both forward and random database searches and FDR ≤1% at the spectrum level, the most protein spectrum matches and protein IDs were obtained at a mass tolerance of 15 ppm. The average mass accuracy of both precursor and product ions from three representative high, medium, and low abundance proteins as well as the common proteins identified in all the 18 replicate runs was found to be 0-4 ppm; and no significant drift of measured mass accuracy was observed within the calibration period of 1 week.
  
  
  
  Despite the mass measurement accuracy of 1-5 ppm of the Orbitrap stated by the manufacturer, the optimal mass tolerance for protein identification was found to be 15 ppm for both the precursor and product ions. Weekly mass calibration is appropriate because no significant drift in MMA was found within the 6-day period. Copyright © 2016 John Wiley & Sons, Ltd.
authors.affiliation: Department of Chemistry and Shanghai Key Laboratory of Chemical Assessment and Sustainability, Tongji University, Shanghai, 200092, China.
authors.forename: Fan
authors.initials: F
authors.lastname: Yu
authors.affiliation: Department of Chemistry and Shanghai Key Laboratory of Chemical Assessment and Sustainability, Tongji University, Shanghai, 200092, China.
authors.forename: Houqin
authors.initials: H
authors.lastname: Fang
authors.affiliation: Department of Chemistry and Shanghai Key Laboratory of Chemical Assessment and Sustainability, Tongji University, Shanghai, 200092, China.
authors.forename: Kaijie
authors.initials: K
authors.lastname: Xiao
authors.affiliation: Department of Chemistry and Shanghai Key Laboratory of Chemical Assessment and Sustainability, Tongji University, Shanghai, 200092, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Chemistry and Shanghai Key Laboratory of Chemical Assessment and Sustainability, Tongji University, Shanghai, 200092, China.
authors.forename: Bingbing
authors.initials: B
authors.lastname: Xue
authors.affiliation: Department of Chemistry and Shanghai Key Laboratory of Chemical Assessment and Sustainability, Tongji University, Shanghai, 200092, China.
authors.forename: Zhixin
authors.initials: Z
authors.lastname: Tian
mesh_terms: D056148:Chromatography, Reverse-Phase; D004926:Escherichia coli; D006801:Humans; D007477:Ions; D020543:Proteome; D053719:Tandem Mass Spectrometry
chemical_list: D007477:Ions; D020543:Proteome