Direct spectrum-centric analysis of diaPASEF data using diaTracer and FragPipe computational platform

Data-independent acquisition (DIA) has become a widely used strategy for peptide and protein quantification in mass spectrometry-based proteomics studies. The integration of ion mobility separation into DIA analysis, such as the diaPASEF technology available on Bruker"s timsTOF platform, further improves the quantification accuracy and protein depth achievable using DIA. However, the complex data presents new challenges for computational analyses. Creating the spectra library built by additional data-dependent acquisition (DDA) experiments requires additional effort. And the in-silico predicted spectral library is limited in the post-translational modification and no-specific searches. An alternative computational strategy, first proposed by us and implemented in the DIA-Umpire algorithm, generates DDA-like pseudo-MS/MS spectra by detecting extracted ion chromatogram (XIC) features directly from DIA data. However, DIA-Umpire cannot process diaPASEF data due to the challenges of detecting XIC features with the additional ion mobility dimension.

To address the need for an efficient and versatile method for analyzing diaPASEF data, we developed diaTracer, a novel direct spectrum-centric tool. diaTracer has been designed to perform highly efficient processing of three-dimensional information (m/z, retention time, ion mobility) to detect precursor and fragment ion features, followed by the generation of pseudo-MS/MS spectra. It is fully integrated into the widely used FragPipe computational platform for direct analysis of diaPASEF data. We demonstrate the performance of diaTracer using diaPASEF data from cerebrospinal fluid (CSF) and plasma proteome datasets, as well as data from phosphoproteomics, immunopeptidomics, and low-input spatial proteomics studies. We also show that our strategy enables unrestricted PTM searches by applying the open/mass offset mode of MSFragger to diaTracer-extracted files. Overall, our preliminary analysis shows that diaTracer, coulpled with FragPipe computational platform, provides a one-stop, fast, and sensitive solution for analyzing of diaPASEF data from a wide range of biological applications.