Spectral Deconvolution of SWATH Data for Peptide Identification and Deciphering HIV-1 Antiviral Response Mechanisms
Mass spectrometry (MS) has become the main analytical technique to characterize proteins and small molecules in complex samples. In proteomics, proteins are digested into peptides and complex peptide mixtures are separated by liquid chromatography (LC). This procedure is followed by electrospray ionization (ESI), resulting in peptide ions that are analyzed by MS.
In this very popular so-called shotgun approach, the mass spectrometer is operated in data-dependent acquisition (DDA) mode, where the top n most intense ions detected in a survey scan are selected for subsequent isolation and fragmentation in a serial manner, generating high-purity spectra.
In contrast to this sequential analysis of individual ions, data-independent acquisition (DIA) methods such as SWATH, systematically parallelizes the fragmentation of all detectable ions within a wide mass range regardless of intensity. They thereby provide increased reproducibility for identification, improved sensitivity and accuracy for quantification and, potentially, enhanced proteome coverage.
Nonetheless, SWATH requires more sophisticated data analysis post-acquisition compared to DDA since convoluted or multiplexed MS/MS spectra are generated without explicit association between each single peptide and its corresponding fragments. At present, SWATH data processing is guided by MS information from preliminary DDA analyses.
In order to by-pass using prior DDA-based MS peptide knowledge, we plan to develop an effective deconvolution algorithm and directly process SWATH data. As a general approach, the algorithm will be applicable to any proteomics study and will open the possibility of identifying proteins undetected in prior MS analyses.
More specifically, we are exploiting the advantages of SWATH within a systems biology approach to understand the role of human dendritic cells (DCs) in the coordination of innate and acquired immune responses during HIV-1 infection and unravel the mechanisms by which DCs determine disease outcome.
Schlagworte: Mass Spectrometry-Based Proteomics, Data-Independent Acquisition, SWATH, Spectral Deconvolution, Spectral Demultiplexing, Peptide Identification, Proteome Mapping, Immune Response, HIV-1 Infectionzurück