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Ion mobility spectrometry with acquisition-based single-shot proteomics

Goutam Rout

Ion mobility spectrometry, a method that divides gas phase ions by their size and shape and may be hybridised with traditional LC and MS, has recently gained popularity thanks to improvements in efficiency and simplicity of use. Here, we examine the most recent advancements in Trapped Ion Mobility Spectrometry (TIMS) in conjunction with TOF mass analysis. Particularly in terms of sequencing speed and sensitivity, the Parallel Accumulation-Serial Fragmentation (PASEF) operation mode offers exceptional benefits. Its distinguishing characteristic is that it coordinates the downstream selection of precursors for fragmentation in a TIMS quadrupole TOF configuration with the release of ions from the TIMS device. When doing data-dependent or focused analysis, the number of peptide fragment ion spectra obtained decreases as ions are compressed into small ion mobility peaks data-dependent or targeted analyses can be increased by an order of magnitude without compromising sensitivity. The PASEF concept increases the effectiveness of data-independent collection by taking advantage of the correlation between ion mobility and mass. This makes the method suitable for ultrasensitive measurements down to single cells as well as quick proteome profiling, a feature that is becoming more and more significant in clinical proteomics. The development of neural networks that can predict collisional cross section values solely based on peptide sequences is made possible by the speed and accuracy of TIMS and PASEF. PASEF also allows for precise measurements of collisional cross section values at the scale of more than a million data points. For isobaric sequences or positional isomers of post-translational modifications, the values of the peptide collisional cross section can vary. This additional data may be used to guide data collecting in real time or to boost the accuracy of peptide identifications after post-processing. Due to these advancements, TIMS quadrupole TOF PASEF is now a strong and flexible platform for proteomics and other fields.