Native
electrospray ionization was known to preserve the protein
structure in solution, which overcame the uncontrollable acidification
of droplets during transfer from solution into the gas phase in conventional
electrospray ionization. However, detailed experimental studies on
when and how could native electrospray ionization minimize structural
perturbations remain quite unclear. Herein, we conducted molecular
dynamics simulations to investigate the protein structure evolution
during electrospray ionization. At a neutral droplet pH, the protein
structure in solution could be retained after evaporation, which was
in accordance with previous reports. As the droplet pH deviated from
neutral, we have found that the compact protein structure would not
unfold until the last 10 ns prior to the final desolvation, which
demonstrated that the role of native electrospray ionization in preserving
the protein structure was mainly reflected on the final evaporation
stages. The present study might provide new insights into studying
the microscopic biomolecular events occurring during the liquid–gas
interface transition and their influence on solution–structure
retention.
Native
mass spectrometry, which takes a high concentration
of ammonium
acetate (NH4OAc) for ionization, coupled with tedious and
solvent-consuming purification, which separates proteins from complicated
environments, has shown great potential for proteins and their complexes.
A high level of nonvolatile salts in the endogenous intracellular
environment results in serious ion suppression and has been one of
the bottlenecks for native mass spectrometry, especially for protein
complexes. Herein, an integrated protocol utilizing the inner surface
of a micropipette for rapid purification, desorption, and ionization
of peptide–metal interaction at subfemtomole level in cell
lysate was demonstrated for native mass spectrometry. The methods
showed robust and reproducibility in protein measurement within 1
min from various buffers. The E. coli cells expressing
with various proteins were lysed and used to test our method. The
specific interaction between the peptide–metal complex in cell
lysates could be reserved and distinguished by mass spectrometry.
The mechanisms whereby protein ions are released from
nanodroplets
at the liquid–gas interface have continued to be controversial
since electrospray ionization (ESI) mass spectrometry was widely applied
in biomolecular structure analysis in solution. Several viable pathways
have been proposed and verified for single-domain proteins. However,
the ESI mechanism of multi-domain proteins with more complicated and
flexible structures remains unclear. Herein, dumbbell-shaped calmodulin
was chosen as a multi-domain protein model to perform molecular dynamics
simulations to investigate the structural evolution during the ESI
process. For [Ca4CAM], the protein followed the classical
charge residue model. As the inter-domain electrostatic repulsion
increased, the droplet was found to split into two sub-droplets, while
stronger-repulsive apo-calmodulin unfolded during the early evaporation
stage. We designated this novel ESI mechanism as the domain repulsion
model, which provides new mechanistic insights into further exploration
of proteins containing more domains. Our results suggest that greater
attention should be paid to the effect of domain–domain interactions
on structure retention during liquid–gas interface transfer
when mass spectrometry is used as the developing technique in gas
phase structural biology.
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