3D-Printed High-Pressure-Resistant Immobilized Enzyme Microreactor (µIMER) for Protein Analysis.

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.

Covalent polyphenol modification of a reactive cysteine in the major apple allergen Mal d 1.

Naturally occurring polyphenols can modify the molecular properties of food allergens. For the major apple allergen Mal d 1 it has been postulated that chemical reactions with polyphenols cause permanent changes in the tertiary structure, causing a loss of conformational IgE epitopes and reducing allergenicity. In our study, we investigated the effect that reactions with oxidized polyphenols have on the structure of Mal d 1 by mass spectrometry and NMR spectroscopy. We showed that a surface-exposed cysteine residue in this allergen spontaneously reacts with oxidized polyphenols under formation of a defined covalent adduct. Chemical modification of Mal d 1 did not destabilize or perturb the three-dimensional fold, nor did it interfere with ligand binding to its internal pocket. A structural model of the chemically modified apple allergen is presented, which reveals that the bound polyphenol partially covers a conformational IgE epitope on the protein surface.

Microdroplet Mass Spectrometry Enables Extremely Accelerated Pepsin Digestion of Proteins.

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.