Stephanie Kaspar-Schoenefeld1, Markus Lubeck1, Adam Rainczuk2, Thomas Kosinski1, Scarlet Koch1, Oliver Raether1, Gary Kruppa1
Institutes
1Bruker Daltonik GmbH, 28359 Bremen, Germany
2Bruker Australia, Melbourne, VIC
Introduction
Data-independent acquisition (DIA) promises reproducible and accurate protein identification and quantification across large sample cohorts by using wide selection windows to ensure that all precursor ions are fragmented in every sample. Ion mobility separation provides an additional dimension for separation of complex proteomics samples, that can also be used for alignment of precursor and fragment information. Here, we combine the PASEF technology (Meier et al., 2018) with a DIA approach and investigate the potential for complex proteomics samples using short gradients.
Methods
An in-house tryptic digest of HeLa was analyzed by coupling an Evosep One system (Evosep Biosystems) online to a trapped ion mobility spectrometry – quadrupole time of flight mass spectrometer (timsTOF Pro, Bruker Daltonics). A dia-PASEF scheme optimized for the short gradient methods has been used for targeting +2 and +3 ions in a three-window method covering an m/z range from 400 to 1000 with a total cycle time of 900ms per dia-PASEF cycle. Data processing was done using Spectronaut 14 (Biognosys).
Results
DIA workflows rely on spectral libraries for the correlation of quantitative data from fragment ion spectra with peptide identifications. We used PASEF in DDA mode and fractionated samples to assemble the resource-specific library using Spectronaut software. The library comprised 8,381 protein groups and 93,301 peptide sequences in ~10h of data acquisition. We applied the hybrid library approach supported in Spectronaut for shorter gradient data by combining the resource-specific library with a project-specific library. This workflow allows keeping retention time precision of the shorter gradients for targeted data extraction. Using the comprehensive libraries, we could identify and quantify on average 5,204 protein groups and 39,936 peptide sequences using 60 SPD method at a 1% FDR.
Conclusion
The dia-PASEF method in combination with short gradients enables high sample throughput without sacrificing depth and quantitative accuracy of analysis.