In microdroplets, rates of chemical or biomolecular reactions can exceed those in the bulk phase by more than a million times. As electrospray ionization-based mass spectrometry (MS) involves the formation of charged microdroplets, reaction acceleration and online MS monitoring of reaction products can readily be performed at the same time. We investigated accelerated enzymatic reactions in microdroplets and focused on the proteolytic enzyme pepsin. Electrosonic spray ionization (ESSI) was utilized for developing the ultrarapid pepsin in-spray digestion of two different proteins, cytochrome c and RocC, at low pH values. The optimization of the protein digestion aimed at achieving maximum sequence coverage for the analyzed proteins. Furthermore, carefully designed control experiments allowed us to unambiguously prove that enzymatic protein cleavage almost exclusively occurs within the spray at a millisecond time scale and not prior to microdroplet generation.
Additive manufacturing
(3D printing) has greatly revolutionized
the way researchers approach certain technical challenges. Despite
its outstanding print quality and resolution, stereolithography (SLA)
printing is cost-effective and relatively accessible. However, applications
involving mass spectrometry (MS) are few due to residual oligomers
and additives leaching from SLA-printed devices that interfere with
MS analyses. We identified the crosslinking agent urethane dimethacrylate
as the main contaminant derived from SLA prints. A stringent washing
and post-curing protocol mitigated sample contamination and rendered
SLA prints suitable for MS hyphenation. Thereafter, SLA printing was
used to produce 360 μm I.D. microcolumn chips with excellent
structural properties. By packing the column with polystyrene microspheres
and covalently immobilizing pepsin, an exceptionally effective microscale
immobilized enzyme reactor (μIMER) was created. Implemented
in an online liquid chromatography-MS/MS setup, the protease microcolumn
enabled reproducible protein digestion and peptide mapping with 100%
sequence coverage obtained for three different recombinant proteins.
Additionally, when assessing the μIMER digestion efficiency
for complex proteome samples, it delivered a 144-fold faster and significantly
more efficient protein digestion compared to 24 h for bulk digestion.
The 3D-printed μIMER withstands remarkably high pressures above
130 bar and retains its activity for several weeks. This versatile
platform will enable researchers to produce tailored polymer-based
enzyme reactors for various applications in analytical chemistry and
beyond.
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