Data-independent acquisition (DIA) mass spectrometry is a next generation proteomic methodology that generates highly reproducible proteome maps of cellular and tissue specimens. DIA is a method of molecular structure determination in which all ions within a selected m/z range are fragmented and analyzed. MS/MS spectra are acquired either by fragmenting all ions that enter the mass spectrometer at a given time (called ‘broadband DIA’) or by sequentially isolating and fragmenting ranges of m/z. DIA is an alternative to data-dependent acquisition (DDA) where a fixed number of precursor ions are isolated and selected for fragmentation. A major strength of DIA-MS is the exceptional reproducibility in protein identification across multiple experiments, as it circumvents the stochastic nature of DDA-MS.

Data analysis is generally challenging for DIA methods as the resulting fragment ion spectra are highly multiplexed. In DIA spectra therefore the direct relation between a precursor ion and its fragment ions is lost since the fragment ions in DIA spectra may potentially result from multiple precursor ions (i.e., any precursor ion present in the m/z range from which the DIA spectrum was derived).