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

Abstract

AbstractData 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.