Exploring the Potential of Data-Independent Acquisition Proteomics Using Untargeted All-Ion Quantitation: Application to Tumor Subtype Diagnosis.

Maximizing the recovery of meaningful biological information can facilitate proteomics-guided early detection and precise treatment of diseases. However, the conventional protein and peptide level targeted quantification of untargeted data independent acquisition (DIA) such as sequential window acquisition of all theoretical spectra (SWATH) is not necessarily descriptive of all information. Untargeted all-ion quantification theoretically could retrieve more features in SWATH digital maps by circumventing the initial identification process but is intrinsically susceptible to errors because of the extreme complexity of proteome samples and the poor selectivity of a single ion. In this study, we optimized and applied the untargeted all-ion quantification of SWATH data to differentiate tumor subtypes. Large peptides and low abundant peptides benefited more from untargeted all-ion quantification. Top-ranked significant ions were linked to their corresponding ion envelops, where multiple correlated ions were used for measurement and only ion envelopes containing at least three ions with consistent intensity ratio were kept as refined differentiating features. Multivariate statistical analysis revealed that for the tested data set, the refined markers discovered by untargeted SWATH analysis showed comparable diagnostic power to protein and peptide markers. Limitations and benefits of the approach are further discussed.