A gas phase fractionation acquisition scheme integrating ion mobility for rapid diaPASEF library generation

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Data independent acquisition (DIA/SWATH) MS is a primary strategy in quantitative proteomics. diaPASEF is a recent adaptation using trapped ion mobility spectrometry (TIMS) to improve selectivity/sensitivity. Complex DIA spectra are typically analyzed with reference to spectral libraries. The best-established method for generating libraries uses offline fractionation to increase depth of coverage. More recently strategies for spectral library generation based on gas phase fractionation (GPF), where a representative sample is injected serially using narrow DIA windows that cover different mass ranges of the complete precursor space, have been introduced that performed comparably to deep offline fractionation-based libraries. We investigated whether an analogous GPF-based approach that accounts for the ion mobility (IM) dimension is useful for the analysis of diaPASEF data. We developed a rapid library generation approach using an IM-GPF acquisition scheme in the m/z versus 1/K0 space requiring seven injections of a representative sample and compared this with libraries generated by direct deconvolution-based analysis of diaPASEF data or by deep offline fractionation. We found that library generation by IM-GPF outperformed direct library generation from diaPASEF and had performance approaching that of the deep library. This establishes the IM-GPF scheme as a pragmatic approach to rapid library generation for analysis of diaPASEF data.

Original languageEnglish
Article number2200038
Number of pages8
Early online date10 Mar 2023
Publication statusEarly online date - 10 Mar 2023


  • Biochemistry
  • Molecular Biology
  • diaPASEF
  • protein–protein interaction
  • protein complex
  • DIA


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