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, 13 (12), 3709-15

Ion Mobility Tandem Mass Spectrometry Enhances Performance of Bottom-Up Proteomics

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Ion Mobility Tandem Mass Spectrometry Enhances Performance of Bottom-Up Proteomics

Dominic Helm et al. Mol Cell Proteomics.

Abstract

One of the limiting factors in determining the sensitivity of tandem mass spectrometry using hybrid quadrupole orthogonal acceleration time-of-flight instruments is the duty cycle of the orthogonal ion injection system. As a consequence, only a fraction of the generated fragment ion beam is collected by the time-of-flight analyzer. Here we describe a method utilizing postfragmentation ion mobility spectrometry of peptide fragment ions in conjunction with mobility time synchronized orthogonal ion injection leading to a substantially improved duty cycle and a concomitant improvement in sensitivity of up to 10-fold for bottom-up proteomic experiments. This enabled the identification of 7500 human proteins within 1 day and 8600 phosphorylation sites within 5 h of LC-MS/MS time. The method also proved powerful for multiplexed quantification experiments using tandem mass tags exemplified by the chemoproteomic interaction analysis of histone deacetylases with Trichostatin A.

Figures

Fig. 1.
Fig. 1.
Synchronization of fragment ion mobility and pusher frequency improves the system performance of a Q-TOF instrument. A, Schematic illustration of operation of conventional DDA (above dashed line) and HD-DDA (below dashed line) on a Q-TOF instrument. B, Tandem mass spectrum of GluFib without (-) and with (+) fragment ion mobility synchronization enabled. The insert shows the 10x magnified tandem mass spectrum of the conventional DDA experiment.
Fig. 2.
Fig. 2.
Performance comparison regarding sensitivity of the HD-DDA and DDA methods. A, Dilution series of a HeLa digest from 1.000 ng to 10 ng on column (each analyzed on a 60-min LC-gradient) via DDA (red) and HD-DDA (blue), illustrating the differences of both methods for peptide and protein identification. B, Comparison of the rate of tandem mass spectrum acquisition (in Hz) across the LC-gradient for DDA (red) and HD-DDA (blue).
Fig. 3.
Fig. 3.
Performance evaluation of the HD-DDA method at varying LC gradient times. A, Triplicate analysis of 1.000 ng of HeLa digest using gradient lengths from 15′ to 360′. B, Quantitative reproducibility of two 60′ gradient replicates based on peptide precursor intensity. C, Characteristics of the number of eluting (peptide) features (dashed line) as well as the rate of identification of eluting features and acquired tandem mass spectra at different LC gradient times. D, Qualitative reproducibility of two 60′ gradient replicates based on the number of identified proteins and peptides.
Fig. 4.
Fig. 4.
Proteome profiles of eight human organs using a total of 24h of LC-MS/MS time per organ.
Fig. 5.
Fig. 5.
Evaluation of the suitability of HD-DDA for PTM analysis and MS/MS based quantification. A, Analysis of IMAC enriched phosphopeptides in two technical replicates using 5 h total analysis time. B, TMT-quantification of the interaction of Trichostatin A with cellular proteins employing a competition binding assay using immobilized SAHA and increasing doses of Trichostatin A.

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