Dual‐Laser Laser‐Induced Liquid Bead Ion Desorption Mass Spectrometry: A New Approach for Adjustable Softness and Higher Sensitivity

Biomechanical investigation of native protein complexes is essential to understand structural features, assembly pathways, and dynamic interactions. As sample complexity increases, current analytical platforms reach performance limits, highlighting the need for improved instrumentation. LILBID‐MS has proven to be a highly suitable native mass spectrometry method for identifying affinities, complexes, stoichiometries, and assembly pathways, even for challenging membrane proteins. In LILBID‐MS, microdroplets containing the sample are irradiated by a mid‐IR laser pulse, leading to the desorption of the sample into the gas phase without additional active charging, preserving native‐like interactions. Ion yield and in‐source dissociation both depend on laser energy, creating a trade‐off between signal intensity and soft conditions. Increasing laser energy results in higher ion counts but also boosts in‐source dissociation; consequently, weak protein interactions are difficult to detect. Here, we present a dual‐laser LILBID‐MS approach, which employs two laser pulses with separately controllable intensities and time delay. This new setup allows optimizing the degree of in‐source dissociation independently of ion count. This increased control provides the option to adapt to sample‐specific requirements, e.g., allowing for soft condition measurements, which were previously inaccessible.