Crosslinking mass spectrometry (XL-MS) has emerged as a powerful tool for investigating protein interactions and complex biological systems. However, its widespread application has been hindered by challenges in data acquisition and analysis, particularly in complex biological samples. We present a comprehensive approach that integrates cutting-edge data acquisition strategies and advanced work-up techniques to enhance the output and feasibility of XL-MS in complex systems biology studies.
Our approach incorporates several innovative methodologies, including High field asymmetric waveform ion mobility spectrometry (FAIMS), fractionation prior to Liquid Chromatography-Mass Spectrometry (LC-MS), and a combination of Data-Dependent Acquisition (DDA) and Data-Independent Acquisition (DIA) approaches. These strategies enable enhanced sensitivity, resolution, and throughput, facilitating the identification and characterization of crosslinked peptides in complex mixtures.
Moreover, we make use of the enrichable crosslinking reagent PhoX, which offers distinct advantages in XL-MS experiments by providing improved selectivity and efficiency in crosslink identification.
Through the integration of these advancements, our work demonstrates a significant enhancement in the capabilities of XL-MS, making it more accessible and practical for investigating intricate protein networks and dynamic interactions within complex biological systems.