In recent years mass spectrometry-based proteomics workflows have become ever more powerful in analysing complex proteomes to great depth. Data-independent acquisition (DIA) approaches are particularly attractive due to their reproducibility and data completeness but also have drawbacks: spectral complexity negatively impacts speed and data analysis, and only a few percent of all incoming ions are isolated for mass analysis – a low sampling efficiency.
As in the alternate DDA (data-dependent acquisition) mode, the addition of ion mobility separation to chromatographic and mass separation should help reduce spectral complexity. Use of a trapped ion mobility spectrometer (TIMS) together with PASEF (parallel accumulation–serial fragmentation) in DDA has also shown a significant increase in sequencing speed, without the loss of sensitivity normally expected in such fast fragmentation cycles.
Meier et al now harness these advantages into a DIA workflow. Modifying a Bruker timsTOF Pro mass spectrometer to accept ‘diaPASEF’ acquisition cycles, and using IonOpticks Aurora Series columns, the resulting workflow achieves deep coverage with high quantitative accuracy from mixed organism samples, with almost 100% sampling of the precursor ion beam. This translates into higher sensitivity, high reproducibility, and quantitative accuracy. The diaPASEF workflow can be further optimized to balance selectivity, sensitivity, and precursor coverage.
Additionally, the authors establish a targeted data extraction workflow by inclusion of the added ion mobility dimension, increasing the specificity for precursor identification.
Importantly, the brief cycle time of this new scan mode is also beneficial for short gradients – especially handy for the high throughput required in large scale biological and clinical studies.
Read the full paper
diaPASEF: parallel accumulation–serial fragmentation combined with data-independent acquisition.
Nat Methods. 17, 30 Nov 2020. doi: https://doi.org/10.1038/s41592-020-00998-0
Meier F, Brunner AD, Frank M, Ha A, Bludau I, Voytik E, Kaspar-Schoenefeld S, Lubeck M, Raether O, Bache N, Aebersold R, Collins BC, Röst HL and Mann M.
Commentary by Jarrod Sandow, PhD.
About the author
Jarrod has a background in biotechnology and completed his PhD at the Institute of Medical and Veterinary Science in Adelaide. He is a co-inventor of IonOpticks’ core technology and is driven towards developing innovative solutions for the global proteomics research community that will enable scientists and clinicians to discover more from their samples to accelerate advances in biological and medical research.