Combining Mass Spectrometry and Electron Microscopy for Enhanced Molecular Imaging
0:00:18 Introduction: The speaker introduces the concept of merging mass spectrometry, specifically electrospray ionization mass spectrometry, with high-resolution microscopy techniques like electron microscopy.
0:00:38 Historical Context: The speaker draws a parallel between the work of Robert Hooke in microscopy and the challenges faced in modern sample preparation.
0:02:53 Challenges in Scanning Probe Microscopy (SPM): SPM, despite achieving atomic resolution, requires ultra-high vacuum conditions and thermal sublimation, limiting its application to biological samples.
0:04:44 Electrospray Ionization as a Solution: Electrospray ionization is presented as a method to generate intact gas-phase ions of biological molecules, overcoming the limitations of SPM.
0:05:34 Controlled Ion Deposition: The speaker explains the precise control over ion deposition achieved through mass filtering, beam focusing, and energy adjustment, allowing for controlled sample preparation.
0:07:15 Application to Glycans: The technique is demonstrated on glycans, showing its ability to image branching and connectivity, overcoming the limitations of traditional mass spectrometry in analyzing these complex molecules.
0:10:55 Atomic Resolution with AFM: Non-contact Atomic Force Microscopy (AFM) with a carbon monoxide tip is shown to achieve atomic resolution on glycans, revealing details of sugar subunits.
0:13:04 Atomic Resolution in Cryo-EM: The speaker highlights the capability of cryo-electron microscopy (cryo-EM) to achieve atomic resolution in both material science and biological applications.
0:14:12 Challenges in Cryo-EM Sample Preparation: The speaker discusses the complexities of cryo-EM sample preparation, particularly the blotting and plunge-freezing process, and proposes native electrospray ion beam deposition as an alternative.
0:16:19 Investigating Gas-Phase vs. Solution Structures: The central question is whether structures observed in the gas phase (mass spectrometry) are representative of those in solution (cryo-EM or crystallography).
0:17:35 Instrument Design: A custom-built instrument is described, combining a modified orbitrap with an ion beam column and a specialized landing system for controlled deposition onto cold TEM grids.
0:18:49 Initial Results: Preliminary data from room-temperature deposition onto amorphous carbon grids show promising results, with the rough shape of beta-galactosidase retained.
0:19:56 Optimizing Ion Beam Conditions: The speaker emphasizes the importance of optimizing ion beam conditions to minimize energy spread and ensure gentle treatment of the particles.
0:22:26 Sample Transfer and Substrate Considerations: A specialized transfer system is described to maintain sample integrity during transfer to the cryo-EM, and the choice of amorphous carbon as a substrate is discussed.
0:24:45 Molecular Dynamics Simulations: Simulations are used to model the collision of proteins with a graphene surface, demonstrating that the gas-phase fold is retained at low collision energies.
0:26:05 Ice Embedding: The ability to grow ice in a controlled manner on the sample is demonstrated, offering a potential matrix for cryo-EM studies.
0:26:47 High-Resolution Structures from Deposited Beta-Galactosidase: 2.5 Angstrom resolution structures of deposited beta-galactosidase are obtained, revealing a slightly more compact structure compared to the solution structure due to dehydration.
0:29:22 Molecular Dynamics Simulations of Dehydration: Simulations show that the removal of water molecules leads to a compaction of the protein structure, consistent with the experimental observations.
0:31:03 Outlook and Future Directions: The speaker presents preliminary results on other proteins, including GroEL, C-reactive protein, and GDH, and discusses ongoing work with membrane proteins.
0:32:46 Conclusion: Electrospray ion beam deposition is presented as a viable technique for cryo-EM sample preparation, offering unique insights into protein structure and the effects of dehydration.