Evaluating the Efficacy of a Novel Library-Free DIA Workflow in Immunopeptidomics

Immunopeptidomics plays a crucial role in understanding the immune system, paving the way for the development of targeted immunotherapies. While the proteomics field has mostly switched from DDA to DIA, DDA is still widely used for immunopeptidomics. DIA is generally only used with the aid of spectral library. Due
to the exponentially larger size of a non-tryptic search space, library-free DIA analyses of immunopeptidomics can be more time consuming and less sensitive. To address these challenges, we improved directDIA™ in Spectronaut^® to make library-free analysis of immunopeptidomics competitive. We evaluated DDA, library-based and library-free DIA approach for their feasibility to extract meaningful biological information from immunopeptidomics samples. With our improvements, library-free DIA identifies more biologically relevant peptides with high confidence.