Poster Presentation 23rd Annual Lorne Proteomics Symposium 2018

Trapped ion mobility spectrometry with parallel accumulation serial fragmentation (PASEF): the new standard for shotgun proteomics (#161)

Adam Rainczuk 1 , Scarlet Beck 2 , Markus Lubeck 2 , Heiner Koch 2 , Florian Meier 3 , Jurgen Cox 3 , Oliver Raether 2 , Matthias Mann 3
  1. Bruker Pty Ltd, Preston VIC 3072, VIC, Australia
  2. Bruker Daltonik GmbH, Bremen, Germany
  3. Max Planck Institute of Biochemistry, Planegg, Germany

Background:  The “Parallel Accumulation - Serial Fragmentation” method (PASEF, Meier et al., JPR 2015, PMID: 26538118) for trapped ion mobility spectrometry (TIMS) coupled to a quadrupole time of flight (QTOF) instrument, has been described with the promise of achieving five to ten times faster data dependent acquisition of fragment ion spectra with improvements in sensitivity. Here we present results showing that the promise of PASEF has been realized.

Material & Methods: The performance of a timsTOF instrument with PASEF for shotgun proteomics has been evaluated by using tryptic digests of human cancer cell lysates (HeLa) spiked or not with an UPS peptide mixture, and separated by 90 min nanoLC gradients. Data were analyzed using DataAnalysis (Bruker), Mascot (, and MaxQuant (Cox group, MPI of Biochemistry) or PEAKS (Bioinformatics Solutions).

Results:  A standard 1.1 second PASEF acquisition cycle, which performs over 120 MS/MS at high sensitivity, identifies over 2900 protein groups from a 12 ng injection of a Hela cell digest separated with a 60 min gradient, and over 4800 protein groups from a 100 ng injection with the same gradient. In parallel, ion mobility separation allows to separate isobaric co-eluted peptides prior to fragmentation, further increasing the ID rate.

Conclusion: By enabling data dependent acquisition at very high speeds with improved sensitivity, the timsTOF Pro with PASEF enables researchers to dig deeper into the proteome, using less sample, yet finding more proteins of biological relevance.