title: Immunodepletion plasma proteomics by tripleTOF 5600 and Orbitrap elite/LTQ-Orbitrap Velos/Q exactive mass spectrometers.
abstract: Plasma proteomic experiments performed rapidly and economically using several of the latest high-resolution mass spectrometers were compared. Four quantitative hyperfractionated plasma proteomics experiments were analyzed in replicates by two AB SCIEX TripleTOF 5600 and three Thermo Scientific Orbitrap (Elite/LTQ-Orbitrap Velos/Q Exactive) instruments. Each experiment compared two iTRAQ isobaric-labeled immunodepleted plasma proteomes, provided as 30 labeled peptide fractions, and 480 LC-MS/MS runs delivered >250 GB of data in 2 months. Several analysis algorithms were compared. At 1% false discovery rate, the relative comparative findings concluded that the Thermo Scientific Q Exactive Mass Spectrometer resulted in the highest number of identified proteins and unique sequences with iTRAQ quantitation. The confidence of iTRAQ fold-change for each protein is dependent on the overall ion statistics (Mascot Protein Score) attainable by each instrument. The benchmarking also suggested how to further improve the mass spectrometry parameters and HPLC conditions. Our findings highlight the special challenges presented by the low abundance peptide ions of iTRAQ plasma proteome because the dynamic range of plasma protein abundance is uniquely high compared with cell lysates, necessitating high instrument sensitivity.
authors.affiliation: Fox Chase Cancer Center, Philadelphia, PA 19111.
authors.forename: Kelly A
authors.initials: KA
authors.lastname: Jones
authors.affiliation: Fox Chase Cancer Center, Philadelphia, PA 19111.
authors.forename: Phillip D
authors.initials: PD
authors.lastname: Kim
authors.affiliation: Fox Chase Cancer Center, Philadelphia, PA 19111.
authors.forename: Bhavinkumar B
authors.initials: BB
authors.lastname: Patel
authors.affiliation: Temple University School of Medicine, Philadelphia, PA 19140.
authors.forename: Steven G
authors.initials: SG
authors.lastname: Kelsen
authors.affiliation: Temple University School of Medicine, Philadelphia, PA 19140.
authors.forename: Alan
authors.initials: A
authors.lastname: Braverman
authors.affiliation: Lincoln University, Lincoln, PA 19352.
authors.forename: Derrick J
authors.initials: DJ
authors.lastname: Swinton
authors.affiliation: Proteomics Shared Resources, Fred Hutchinson Cancer Research Center, Seattle, WA 98109.
authors.forename: Philip R
authors.initials: PR
authors.lastname: Gafken
authors.affiliation: Proteomics Shared Resources, Fred Hutchinson Cancer Research Center, Seattle, WA 98109.
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Jones
authors.affiliation: Mass Spectrometry and Proteomics Resource Laboratory, Harvard University, Cambridge, MA 02138.
authors.forename: William S
authors.initials: WS
authors.lastname: Lane
authors.affiliation: Mass Spectrometry and Proteomics Resource Laboratory, Harvard University, Cambridge, MA 02138.
authors.forename: John M
authors.initials: JM
authors.lastname: Neveu
authors.affiliation: Mass Spectrometry and Proteomics Core Facility, Baylor College of Medicine, Houston, TX 77030.
authors.forename: Hon-Chiu E
authors.initials: HE
authors.lastname: Leung
authors.affiliation: Proteomics and Mass Spectrometry Facility, University of Massachusetts Medical School, Worcester, MA 01545.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Shaffer
authors.affiliation: Proteomics and Mass Spectrometry Facility, University of Massachusetts Medical School, Worcester, MA 01545.
authors.forename: John D
authors.initials: JD
authors.lastname: Leszyk
authors.affiliation: Mass Spectrometry Core, Penn State College of Medicine, Hershey, PA 17033.
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Stanley
authors.affiliation: Mass Spectrometry Core, Penn State College of Medicine, Hershey, PA 17033.
authors.forename: Todd E
authors.initials: TE
authors.lastname: Fox
authors.affiliation: Mass Spectrometry Core, Penn State College of Medicine, Hershey, PA 17033.
authors.forename: Anne
authors.initials: A
authors.lastname: Stanley
authors.affiliation: Fox Chase Cancer Center, Philadelphia, PA 19111.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Hall
authors.affiliation: Human Cancer Genetics Program, the Ohio State University, Columbus, OH 43210.
authors.forename: Heather
authors.initials: H
authors.lastname: Hampel
authors.affiliation: Human Cancer Genetics Program, the Ohio State University, Columbus, OH 43210.
authors.forename: Christopher D
authors.initials: CD
authors.lastname: South
authors.affiliation: Human Cancer Genetics Program, the Ohio State University, Columbus, OH 43210.
authors.forename: Albert
authors.initials: A
authors.lastname: de la Chapelle
authors.affiliation: Huntsman Cancer Institute, the U. of Utah, Salt Lake City, UT 84112.
authors.forename: Randall W
authors.initials: RW
authors.lastname: Burt
authors.affiliation: Huntsman Cancer Institute, the U. of Utah, Salt Lake City, UT 84112.
authors.forename: David A
authors.initials: DA
authors.lastname: Jones
authors.affiliation: Division of Cancer Prevention, National Cancer Institute, Bethesda, MD 20892.
authors.forename: Levy
authors.initials: L
authors.lastname: Kopelovich
authors.affiliation: Fox Chase Cancer Center, Philadelphia, PA 19111.
authors.forename: Anthony T
authors.initials: AT
authors.lastname: Yeung
mesh_terms: D001798:Blood Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D010449:Peptide Mapping; D040901:Proteomics; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods* / Q000592:standards
chemical_list: D001798:Blood Proteins