Data Dependent Acquisition (DDA) is the standard method for running LC-MS/MS experiments. Here, we present a quick overview of the DDA method, as well as of the less common Data Independent methods (DIA).
In LC-MS/MS, purified peptides are separated on an online chromatography gradient (usually low pH Reverse Phase) over 1 to 4h and progressively injected as they elute into the Mass Spectrometer by Electrospray Ionisation (ESI).
The standard MS acquisition method is called Data Dependent Acquisition (DDA): in each duty cycle (~1s), the instrument cycles through first a short MS1 survey scan of currently eluting peptides – which serves to monitor peptide intensity and to identify potential targets to fragment – then a series of n (~10) MS2 scans – during each of which a precursor is isolated, fragmented and its product ions detected. Precursors are fragmented in order of decreasing intensity.
A dynamic exclusion window is used to make sure peptides which have recently been analysed by MS2 are not being constantly re-fragmented if there are newer targets available.
Since each peptide will elute over 30-40s, the MS predicts when a peptide it just started detecting is going to peak and will aim to sequence it at maximum abundance to increase the chance of producing a high quality MS2 spectrum.
In recent years, Data Dependent Methods (DIA) have become more frequently used. In DIA, such as SWATHE or MSE, no precursors are isolated, instead all eluted peptides are fragmented. This generates highly complex MS2 spectra which can then be analysed using a spectrum library. While DIA data analysis is more complex, each acquisition has the potential to provide a much more complete picture of the sample, and results can improve as libraries are expanded.