SUMMARY:
• Cantilever based biosensors are a very common type of microsensor
• They are relatively easy to produce and use
• Microdispensing is very useful to activate their surface and functionalize it with different biomolecules
Background
A key challenge that mass spectrometry can address is the investigation of biomolecular structures and interactions. This requires minimally disruptive techniques to enable the study of dynamic processes such as protein complex formation, membrane transporter mechanisms, conformational changes in signaling proteins, and the analysis of non-covalent biomolecular assemblies. To meet this need, the LILBID-MS technology was developed [1] and is now widely used to probe structural rearrangements, binding affinities, and assembly pathways of proteins and complexes in diverse environments including nanodiscs, micelles, and cell-free systems. These insights contribute significantly to our understanding of molecular biology at both structural and mechanistic levels.
The images featured here originate from the research of Prof. Morgner’s group at Goethe University Frankfurt. You can explore more about their work on their website.
Reasons for Using Microdispensing
Microdispensing offers several advantages:
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Minimizes liquid volumes, conserving samples.
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Scalable to different protein concentrations in using anywhere from a few to a few hundred droplets
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Compatible with a broad range of samples , dissolved in aqueous, buffered solution
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Electrically actuated to dispense droplets precisely when needed.
LILBID-MS Process
The LILBID method utilizes microdroplets (50 μm in diameter or smaller), generated by a microdispenser and introduced into a vacuum. These droplets are irradiated with a mid-infrared laser, causing gentle disruption and releasing biomolecular complex ions. These ions are then analyzed using a time-of-flight mass spectrometer. The laser intensity determines the extent of disruption, enabling the study of various protein complexes. Depending on the laser conditions, intact complexes, subcomplexes, or individual subunits can be investigated.
Utility and Outlook
As protein structures gain increasing relevance and structural biology continues to emerge as a key field of the future, LILBID-MS is likely to remain an important method for studying protein–protein interactions.
References
This technique has revealed important insights in:
- Investigating how membrane proteins form oligomeric complexes.
- Studying the stoichiometry of ATP-dependent molecular mechanisms.
- Analyzing the structure of the phage lysis protein MS2-L and its interaction with DnaJ
- Examining the structural rearrangement of amyloid-β.
- Investigating the structure of Mycobacterium lipid transporters.
- Studying different conformations of mitochondrial receptors.
- Exploring the self-assembly of purified F1 subunits from Bacterial F-type ATPase