Data Independent Acquisition (DIA) strategies acquire MS/MS scans systematically and independent of precursor information. Unlike targeted parallel reaction monitoring (PRM) and selected reaction monitoring (SRM) strategies, no prior knowledge of target peptides/proteins is required for DIA and thus requires less investment in assay development compared to targeted proteomics applications.
There are various strategies how to acquire and analyze DIA data.
In principal data is acquired by isolating and fragmenting a certain m/z mass windows and acquiring all the fragment ions in one scan.
Typically the isolation windows are selected to cover the tryptic peptide m/z range in certain time frame.
There are many factors to be considered, like instrument type, scan speed, chromatography and data analysis tools.
Depending on the isolated m/z range the fragmentation spectra are chimera of multiple precursor ions and
the increased MS/MS spectra complexity has been a major challenge for DIA data analysis tools.
Targeted approaches like PRM and SRM analysis are limited to only a handful of peptides without acquisition scheduling or tens to hundreds of peptides per LCMS run with scheduling. DIA on the other hand allows the measurement of much larger numbers of peptides (thousands or even whole proteomes).
Figure 1: DIA acquisition strategy. Precursor ions are isolated in small isolation windows (typically 5-20 m/z) and fragmented. Fragment ions are analyzed in MS/MS scans spanning the full m/z range. Total Cycle Time is the length of time it takes to go through all the isolation windows and cover the m/z range. Note: ion injection (Fill or ion time) happens in parallel with Orbitrap MS/MS fragment detection, to achieve the fastest cycle time, the max fill time should not exceed the Orbitrap scan time. Full MS scans between the DIA MS/MS scans are optional.