Data-independent Acquisition for the orbitrap. A tutorial.

Data-independent acquisition (DIA) is a powerful mass spectrometric technique to perform both protein identification and quantification of complex protein samples. Setting up DIA methods on Orbitrap analyzers requires a thorough overview over the actions the Orbitrap mass spectrometers carry out. This tutorial is written with the intention to give an overview over the important parameters to consider as well as which measurements to carry out to get the most out of your DIA method when setting it up on an Orbitrap mass analyzer. Instead of giving the optimal DIA settings, all steps in the construction and optimization of the DIA method are shown and discussed in a way that allow tailored DIA methods. Key steps are: building the spectral library after sample fractionation, decide upon the number of data points per chromatographic peak, determine scan times of each mass spectrometric step, construct various DIA methods using these data and evaluate their performance. This proposed DIA method development strategy was tested on digested lysates from Pseudomonas Aeruginosa and compared to conventional DDA analysis to put the DIA results in a perspective.