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Home arrow Health arrow Analysis of Protein Post-Translational Modifications by Mass Spectrometry

Data-Independent Acquisition MS

Data-independent acquisition (DIA) offers the combined benefits of SRM (targeted) and DDA (discovery) MS modes. In this approach, all precursors within a given retention time and m/z window MS1 scan are subjected to a “fragment all” MS2 fragmentation. DDA results in the fragmentation of individual peptides triggered based on an intensity threshold, which occurs on a stochastic basis and thus does not acquire data for all peptides. DIA is distinct from DDA in acquiring all precursor and fragment signals from all peptides across a sample set. As such, DIA provides a comprehensive peptide profile. DIA data can be mined for targeted extraction of both qualitative (PTM diagnostic, isomer-specific ions) and quantitative (intensity) information using DDA-derived spectral libraries containing retention time and fragmentation information for the desired peptide species. DIA spectral libraries can be derived by the alignment of precursor and fragment chromatographic features to form pseudo tandem mass spectra [64]. A major benefit of DIA techniques, in general, is that the data can be interrogated at a later stage, for example, for additional/ new PTMs. Quantitative information can be derived at the level of both precursor and product ions, from the MS1 and MS2 scans [65].

DIA mode of acquisition is available on quadrupole-orbitrap (all-ion fragmentation) and quadrupole TOF MS (SWATH, MSE). All-ion HCD fragmentation is performed on Q Exactive MS instrumentation alternating MS and “all-ion fragmentation” MS2 scans [66]. In MSE, all ions are transferred to the collision cell for CID, intact peptides are measured in the low-energy scans, whereas fragment ions are measured in the high-energy scans, which cycle throughout the run [67]. In SWATH acquisition, the MS scans through the entire mass range of precursors in user-defined window sizes; all precursor ions in each SWATH window are simultaneously fragmented. The MS1 survey scan for the full mass range (shorter acquisition time relative to DDA) is followed by a SWATH MS2 acquisition series. The mass window is typically <25 Da, but these scan accumulation times can be adjusted based on m/z distribution through the LC run to enhance the sensitivity of the analysis, based on MS1 intensity [65]. In general, DIA MS1 precursor ions can be subject to interference, which can be minimized by the reduction of the MS1 window size. MS2 ions are more selective and thus subject to less interference and thus have superior linear response and dynamic range relative to MS1 [65]. A specific benefit of DIA methodology is the ability to resolve and quantify isobaric H3 peptides, for example, the Kac18 and Kac23 peptides [68].

DIA is of particular value for acetylated and/or methylated peptides lacking unique b and y type transition ions. These are refractory to SRM/MRM/PRM since they cannot be discriminated. In DIA the intensity values of precursor and fragments (MS1 and MS2 signals, respectively) can be analyzed to calculate the contributions of individual peptides to the overall abundance of a given set of coeluting isobaric peptides [69]. To do this, the average areas of the chimeric precursor intensities are extracted, and where possible, each peptide of the subset is quantified on the basis of unique sets of b and y ions. Where no unique b or y ions exist, the relative contribution of those peptides is calculated as the proportion of the total remaining. This makes the assumption that ionization efficiencies are similar for the peptide set, which was demonstrated to be appropriate in this study but might not generally translate to other protein PTM peptides. Interestingly, fold change values were strongly correlated between MS1 and MS2 indicating that these values can be used either alone or combined for accurate analysis. Profiling of 62 histone H3 and H4 protein PTMs monitored changes in methylation and acetylation in response to the HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA). DIA acquisition was performed on a Q Exactive MS with MS1 scan in the range 390-910 m/z, and DIA was performed by dividing the m/z range into smaller m/z ranges for sequential isolation (10 m/z units) and fragmentation, using a defined list of target m/z values for specificity [69].

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