A protocol for studying structural dynamics of proteins by quantitative crosslinking mass spectrometry and data-independent acquisition

Quantitative crosslinking mass spectrometry (QCLMS) reveals structural details of protein conformations in solution. QCLMS can benefit from data-independent acquisition (DIA), which maximises accuracy, reproducibility and throughput of the approach. This DIA-QCLMS protocol comprises of three main sections: sample preparation, spectral library generation and quantitation. The DIA-QCLMS workflow supports isotope-labelling as well as label-free quantitation strategies, uses xiSEARCH for crosslink identification, and xiDIA-Library to create a spectral library for a peptide-centric quantitative approach. We integrated Spectronaut, a leading quantitation software, to analyse DIA data. Spectronaut supports DIA-QCLMS data to quantify crosslinks. It can be used to reveal the structural dynamics of proteins and protein complexes, even against a complex background. In combination with photoactivatable crosslinkers (photo-DIA-QCLMS), the workflow can increase data density and better capture protein dynamics due to short reaction times. Additionally, this can reveal conformational changes caused by environmental influences that would otherwise affect crosslinking itself, such as changing pH conditions.