Ultrafast enzymatic digestion of proteins by microdroplet mass spectrometry.
Reads0
Chats0
TLDR
The use of aqueous microdroplets to accelerate enzymatic reactions and, in particular, to improve protein sequencing is reported, achieving full protein sequence coverage in less than 1 ms by subjecting protein-protease mixtures to electrosonic spray ionization-MS.Abstract:
Enzymatic digestion for protein sequencing usually requires much time, and does not always result in high sequence coverage. Here we report the use of aqueous microdroplets to accelerate enzymatic reactions and, in particular, to improve protein sequencing. When a room temperature aqueous solution containing 10 µM myoglobin and 5 µg mL−1 trypsin is electrosonically sprayed (−3 kV) from a homemade setup to produce tiny (∼9 µm) microdroplets, we obtain 100% sequence coverage in less than 1 ms of digestion time, in sharp contrast to 60% coverage achieved by incubating the same solution at 37 °C for 14 h followed by analysis with a commercial electrospray ionization source that produces larger (∼60 µm) droplets. We also confirm the sequence of the therapeutic antibody trastuzumab (∼148 kDa), with a sequence coverage of 100% for light chains and 85% for heavy chains, demonstrating the practical utility of microdroplets in drug development. Mass spectrometry (MS)-based protein sequencing usually relies on in-solution proteolytic digestion, which is time-consuming and inefficient for certain proteins. Here, the authors achieve full protein sequence coverage in less than 1 ms by subjecting protein-protease mixtures to electrosonic spray ionization-MS.read more
Citations
More filters
Journal ArticleDOI
A critical analysis of electrospray techniques for the determination of accelerated rates and mechanisms of chemical reactions in droplets
Grazia Rovelli,Michael I. Jacobs,Michael I. Jacobs,Megan D. Willis,Rebecca J. Rapf,Alexander M. Prophet,Alexander M. Prophet,Kevin R. Wilson +7 more
TL;DR: The limitations of these approaches and the experimental best practices required to robustly interpret acceleration factors in micro- and nano-droplets produced by ESI and ESSI are assessed.
Journal ArticleDOI
Accelerated reactions of amines with carbon dioxide driven by superacid at the microdroplet interface
TL;DR: Electrospray ionization mass spectrometry displays the reaction products in the form of the protonated and deprotonated carbamic acid and trace amounts of water accelerate the reaction, presumably by formation of superacid or superbase at the water interface.
Journal ArticleDOI
Spontaneous Reduction-Induced Degradation of Viologen Compounds in Water Microdroplets and Its Inhibition by Host-Guest Complexation.
TL;DR: In this paper , the authors present the results of the spontaneous and ultrafast reduction-induced degradation of three viologen compounds in water microdroplets and provide the concentration, time, temperature dependence, mechanism, and scale-up of the reactions.
Journal ArticleDOI
Reaction Acceleration Promoted by Partial Solvation at the Gas/Solution Interface
TL;DR: The kinetics of organic reactions of different types in microvolumes (droplets, thin films, and sealed tubes) show effects of gas/solution interfacial area, reaction molecularity and solvent polarity as discussed by the authors.
Journal ArticleDOI
Electrochemical quantification of accelerated FADGDH rates in aqueous nanodroplets
TL;DR: In this paper, the authors demonstrate rate enhancement of up to two orders of magnitude for enzymes trapped in submicrometer water nanodroplets suspended in 1,2-dichloroethane.
References
More filters
Journal ArticleDOI
Structural absorption by barbule microstructures of super black bird of paradise feathers
TL;DR: Physical structure is known to contribute to the appearance of bird plumage through structural color and specular reflection, but a third mechanism, structural absorption, leads to low reflectance and super black color in birds of paradise feathers.
Journal ArticleDOI
In-gel digestion for mass spectrometric characterization of proteins and proteomes
TL;DR: This protocol is for the in-gel digestion of both silver and Coomassie-stained protein spots or bands and can be followed by MALDI-MS or LC-MS/MS analysis to identify proteins at sensitivities better than a few femtomoles of protein starting material.
Journal ArticleDOI
Protein Analysis by Shotgun/Bottom-up Proteomics
TL;DR: The progress of proteomics has been driven by the development of new technologies for peptide/protein separation, mass spectrometry analysis, isotope labeling for quantification, and bioinformatics data analysis.
Journal ArticleDOI
From ions in solution to ions in the gas phase - the mechanism of electrospray mass spectrometry
Paul Kebarle,Liang. Tang +1 more
Journal ArticleDOI
Charge and fission of droplets in electrostatic sprays
Alessandro Gomez,Keqi Tang +1 more
TL;DR: The Coulomb fission phenomenon was also captured in microphotographs that typically showed a droplet with one or two diametrically opposed conical protrusions terminating in a fine jet ejecting a stream of much smaller, apparently equisized offsprings as discussed by the authors.