Abstract
Ion mobility brings an additional dimension of separation to liquid chromatography-mass spectrometry, improving identification of peptides and proteins in complex mixtures. A recently introduced timsTOF mass spectrometer (Bruker) couples trapped ion mobility separation to time-of-flight mass analysis. With the parallel accumulation serial fragmentation (PASEF) method, the timsTOF platform achieves promising results, yet analysis of the data generated on this platform represents a major bottleneck. Currently, MaxQuant and PEAKS are most commonly used to analyze these data. However, due to the high complexity of timsTOF PASEF data, both require substantial time to perform even standard tryptic searches. Advanced searches (e.g. with many variable modifications, semi- or non-enzymatic searches, or open searches for post-translational modification discovery) are practically impossible. We have extended our fast peptide identification tool MSFragger to support timsTOF PASEF data, and developed a label-free quantification tool, IonQuant, for fast and accurate 4D feature extraction and quantification. Using HeLa dataset published by Meier et al. (2018), we demonstrate that MSFragger identifies significantly (∼30%) more unique peptides than MaxQuant (1.6.10.43), and performs comparably or better than PEAKS X+ (∼10% more peptides). IonQuant outperforms both in terms of number of quantified proteins while maintaining good quantification accuracy. Runtime tests show that MSFragger and IonQuant can fully process a typical two hour PASEF run in under 50 minutes on a modern desktop (6 CPU cores, 32 GB RAM), significantly faster than other tools. Finally, through semi-tryptic searching, we annotate significantly (63%) more peptides. Within these semi-tryptic identifications, we report evidence of gas-phase fragmentation prior to MS/MS analysis.
Abbreviations
- LC-MS
- liquid chromatography-mass spectrometry
- IM
- ion mobility
- TIMS
- trapped ion mobility spectrometry
- TOF
- time-of-flight
- PASEF
- parallel accumulation-serial fragmentation
- DDA
- data-dependent acquisition
- DIA
- data-independent acquisition
- MS/MS
- tandem mass spectrometry
- PSM
- peptide-spectrum match
- LC-IMS-MS
- liquid chromatography-ion mobility-mass spectrometry
- XIC
- extracted ion chromatogram
- CV
- coefficient of variation
- LFQ
- label free quantification
- FDR
- False Discovery Rate
- CID
- collision-induced dissociation
- ISF
- In-source fragmentation
- PDV
- proteomics data viewer
- CPU
- central processing unit
- PTM
- post-translational modification