A Novel Differential Ion Mobility Device Expands the Depth of Proteome Coverage and the Sensitivity of Multiplex Proteomic Measurements

The depth of proteomic analyses is often limited by the overwhelming proportion of confounding background ions that compromise the identification and quantification of low abundance peptides. To alleviate these limitations, we present a new high field asymmetric waveform ion mobility spectrometry (FAIMS) interface that can be coupled to the Orbitrap Tribrid mass spectrometers. The interface provides several advantages over previous generations of FAIMS devices, including ease of operation, robustness, and high ion transmission. Replicate LC-FAIMS-MS/MS analyses (n = 100) of HEK293 protein digests showed stable ion current over extended time periods with uniform peptide identification on more than 10,000 distinct peptides. For complex tryptic digest analyses, the coupling of FAIMS to LC-MS/MS enabled a 30% gain in unique peptide identification compared with non-FAIMS experiments. Improvement in sensitivity facilitated the identification of low abundance peptides, and extended the limit of detection by almost an order of magnitude. The reduction in chimeric MS/MS spectra using FAIMS also improved the precision and the number of quantifiable peptides when using isobaric labeling with tandem mass tag (TMT) 10-plex reagent. We compared quantitative proteomic measurements for LC-MS/MS analyses performed using synchronous precursor selection (SPS) and LC-FAIMS-MS/MS to profile the temporal changes in protein abundance of HEK293 cells following heat shock for periods up to 9 h. FAIMS provided 2.5-fold increase in the number of quantifiable peptides compared with non-FAIMS experiments (30,848 peptides from 2,646 proteins for FAIMS versus 12,400 peptides from 1,229 proteins with SPS). Altogether, the enhancement in ion transmission and duty cycle of the new FAIMS interface extended the depth and comprehensiveness of proteomic analyses and improved the precision of quantitative measurements. The depth of proteomic analyses is often limited by the overwhelming proportion of confounding background ions that compromise the identification and quantification of low abundance peptides. To alleviate these limitations, we present a new high field asymmetric waveform ion mobility spectrometry (FAIMS) interface that can be coupled to the Orbitrap Tribrid mass spectrometers. The interface provides several advantages over previous generations of FAIMS devices, including ease of operation, robustness, and high ion transmission. Replicate LC-FAIMS-MS/MS analyses (n = 100) of HEK293 protein digests showed stable ion current over extended time periods with uniform peptide identification on more than 10,000 distinct peptides. For complex tryptic digest analyses, the coupling of FAIMS to LC-MS/MS enabled a 30% gain in unique peptide identification compared with non-FAIMS experiments. Improvement in sensitivity facilitated the identification of low abundance peptides, and extended the limit of detection by almost an order of magnitude. The reduction in chimeric MS/MS spectra using FAIMS also improved the precision and the number of quantifiable peptides when using isobaric labeling with tandem mass tag (TMT) 10-plex reagent. We compared quantitative proteomic measurements for LC-MS/MS analyses performed using synchronous precursor selection (SPS) and LC-FAIMS-MS/MS to profile the temporal changes in protein abundance of HEK293 cells following heat shock for periods up to 9 h. FAIMS provided 2.5-fold increase in the number of quantifiable peptides compared with non-FAIMS experiments (30,848 peptides from 2,646 proteins for FAIMS versus 12,400 peptides from 1,229 proteins with SPS). Altogether, the enhancement in ion transmission and duty cycle of the new FAIMS interface extended the depth and comprehensiveness of proteomic analyses and improved the precision of quantitative measurements. Over the past decade, significant improvements in sensitivity, resolution, and scan speed of mass spectrometers have extended the depth of proteomic analyses. Peptide identification by mass spectrometry (MS) relies on the detection of precursor ions with predefined charge state and their sequential selection for fragmentation as they elute from the chromatographic column (1Beck S. Michalski A. Raether O. Lubeck M. Kaspar S. Goedecke N. Baessmann C. Hornburg D. Meier F. Paron I. Kulak N.A. Cox J. Mann M. The Impact 2 a very high resolution qTOF for deep shotgun proteomics.Mol. Cell. Proteomics. 2015; 14: 2014-2029Abstract S. A. of the Orbitrap mass spectrometry 2015; For low of ion for precursor ion proteomic analyses a in of the overwhelming peptide the of chimeric tandem mass spectra from isobaric ions and the of low abundance peptide ions that often with background the of peptide identification and the depth of analyses can also compromise quantitative measurements. For the precursor ion be in and on the MS/MS spectra to the peptide spectra the selection of the precursor ion of the to the of MS/MS spectra on ions in the can be using and that on peptide J. Paron I. N. Mann M. quantification by and peptide Cell. Proteomics. as and labeling that on the of peptide from the scan can be by the of confounding ions that ion and the of ions of can also the and precision of quantitative when using isobaric peptide labeling the and of ions in ion from isobaric of the peptide and ions that be from N.A. and precision in Cell. Proteomics. M. J. C. I. in and complex the and the The resolution of ions often ion fragmentation and synchronous precursor selection I. the of using ion with isobaric M. and detection of To and alleviate with several on peptide improved resolution, and For C. S. shotgun for the of A. M. and deep by on a Orbitrap mass and column M. N. N. by using column with A. N. Kulak N.A. Cox J. N. O. O. Mann M. with of the by on a Cell. Proteomics. to for analyses. in resolution and have enabled more and transmission of ions with in chimeric tandem mass spectra O. Hornburg D. A. A. Mann M. The a mass with a and an Orbitrap Cell. Proteomics. of that high resolution of scan with of MS/MS the that and peptide ions A. Cox J. Mann M. than peptide elute in shotgun the is to more than The resolution of also the of isobaric ions with charge that their selection for MS/MS The of that can and several in the and can peptide identification by more than in a C. A. S. precursor for mass can with for quantitative using isobaric peptide labeling is by the of chimeric MS/MS spectra and often have also the depth of that in as we sensitivity and peptide ions of improvements in resolution and have the of peptide the proportion of and quantifiable peptides A. Cox J. Mann M. than peptide elute in shotgun the is to is for ion the ion is to for ion and S. of on protein quantification isobaric enhancement of that can the chromatographic of current the identification of peptides over several of in ion using ion mobility spectrometry new to of proteomic a of of to the of peptides and including the of protein I. on ion mobility and of 2015; C. F. mobility spectrometry of and to F. mobility spectrometry and asymmetric waveform ion mobility spectrometry for mass Proteomics. 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