Immunodepletion Plasma Proteomics by TripleTOF 5600 and Orbitrap Elite/LTQ-Orbitrap Velos/Q Exactive Mass Spectrometers

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.