Immunodepletion plasma proteomics by tripleTOF 5600 and Orbitrap elite/LTQ-Orbitrap Velos/Q exactive mass spectrometers
Department of Biochemistry and Molecular Pharmacology; Proteomics and Mass Spectrometry Facility
Medical Subject Headings
Blood Proteins; Humans; Immunoprecipitation; Peptide Mapping; Proteomics; Reference Standards; Reproducibility of Results; Sensitivity and Specificity; Tandem Mass Spectrometry
Biochemistry, Biophysics, and Structural Biology | Chemistry
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.
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Citation: J Proteome Res. 2013 Oct 4;12(10):4351-65. doi: 10.1021/pr400307u. Epub 2013 Sep 19. Link to article on publisher's site
Jones, Kelly A.; Kim, Phillip D.; Shaffer, Scott A.; Leszyk, John D.; and Yeung, Anthony T., "Immunodepletion plasma proteomics by tripleTOF 5600 and Orbitrap elite/LTQ-Orbitrap Velos/Q exactive mass spectrometers" (2013). University of Massachusetts Medical School Faculty Publications. 764.