Mag-Net: Rapid enrichment of membrane-bound particles enables high coverage quantitative analysis of the plasma proteome.

Membrane-bound particles in plasma are composed of exosomes, microvesicles, and apoptotic bodies and represent ~1-2% of the total protein composition. Proteomic interrogation of this subset of plasma proteins augments the representation of tissue-specific proteins, representing a "liquid biopsy," while enabling the detection of proteins that would otherwise be beyond the dynamic range of liquid chromatography-tandem mass spectrometry of unfractionated plasma. We have developed an enrichment strategy (Mag-Net) using hyper-porous strong-anion exchange magnetic microparticles to sieve membrane-bound particles from plasma.

Protocol for high-throughput semi-automated label-free- or TMT-based phosphoproteome profiling.

Tandem mass tags data-dependent acquisition (TMT-DDA) as well as data-independent acquisition-based label-free quantification (LFQ-DIA) have become the leading workflows to achieve deep proteome and phosphoproteome profiles. We present a modular pipeline for TMT-DDA and LFQ-DIA that integrates steps to perform scalable phosphoproteome profiling, including protein lysate extraction, clean-up, digestion, phosphopeptide enrichment, and TMT-labeling. We also detail peptide and/or phosphopeptide fractionation and pre-mass spectrometry desalting and provide researchers guidance on choosing the best workflow based on sample number and input.

Zirconium(IV)-IMAC Revisited: Improved Performance and Phosphoproteome Coverage by Magnetic Microparticles for Phosphopeptide Affinity Enrichment.

Phosphopeptide enrichment is an essential step in large-scale, quantitative phosphoproteomics by mass spectrometry. Several phosphopeptide affinity enrichment techniques exist, such as immobilized metal-ion affinity chromatography (IMAC) and metal oxide affinity chromatography (MOAC). We compared zirconium(IV) IMAC (Zr-IMAC) magnetic microparticles to more commonly used titanium(IV) IMAC (Ti-IMAC) and TiO magnetic microparticles for phosphopeptide enrichment from simple and complex protein samples prior to phosphopeptide sequencing and characterization by mass spectrometry (liquid chromatography-tandem mass spectrometry, LC-MS/MS).

Sustainable production of nucleoside analogues by a high-efficient purine 2'-deoxyribosyltransferase immobilized onto Ni chelate magnetic microparticles.

The present work aims to develop a magnetic biocatalyst for customized production of nucleoside analogues using mutant His-tagged purine 2'-deoxyribosyltransferase from Trypanosoma brucei (TbPDT) immobilized onto Ni chelate magnetic iron oxide porous microparticles (MTbPDT). Biochemical characterization revealed MTbPDT5 as optimal candidate for further studies (10,552 IU g; retained activity 54% at 50 °C and pH 6.5).

The co-immobilization of P450-type nitric oxide reductase and glucose dehydrogenase for the continuous reduction of nitric oxide via cofactor recycling.

The co-immobilization of enzymes on target surfaces facilitates the development of self-contained, multi-enzyme biocatalytic platforms. This generally entails the co-immobilization of an enzyme with catalytic value in combination with another enzyme that performs a complementary function, such as the recycling of a critical cofactor. In this study, we co-immobilized two enzymes from different biological sources for the continuous reduction of nitric oxide, using epoxide- and carboxyl-functionalized hyper-porous microspheres.

Spectrophotometric activity microassay for pure and recombinant cytochrome P450-type nitric oxide reductase.

Nitric oxide reductase (NOR) of the P450 oxidoreductase family accepts electrons directly from its cofactor, NADH, to reduce two nitric oxide (NO) molecules to one nitrous oxide molecule and water. The enzyme plays a key role in the removal of radical NO produced during respiratory metabolism, and applications in bioremediation and biocatalysis have been identified. However, a rapid, accurate, and sensitive enzyme assay has not yet been developed for this enzyme family.

Immobilisation and characterisation of biocatalytic co-factor recycling enzymes, glucose dehydrogenase and NADH oxidase, on aldehyde functional ReSyn™ polymer microspheres.

The use of enzymes in industrial applications is limited by their instability, cost and difficulty in their recovery and re-use. Immobilisation is a technique which has been shown to alleviate these limitations in biocatalysis. Here we describe the immobilisation of two biocatalytically relevant co-factor recycling enzymes, glucose dehydrogenase (GDH) and NADH oxidase (NOD) on aldehyde functional ReSyn™ polymer microspheres with varying functional group densities.

Advances in enzyme immobilisation.

Improvements in current strategies for carrier-based immobilisation have been developed using hetero-functionalised supports that enhance the binding efficacy and stability through multipoint attachment. New commercial resins (Sepabeads) exhibit improved protein binding capacity. Novel methods of enzyme self-immobilisation have been developed (CLEC, CLEA, Spherezyme), as well as carrier materials (Dendrispheres), encapsulation (PEI Microspheres), and entrapment.

Spherezymes: a novel structured self-immobilisation enzyme technology.

Background: Enzymes have found extensive and growing application in the field of chemical organic synthesis and resolution of chiral intermediates. In order to stabilise the enzymes and to facilitate their recovery and recycle, they are frequently immobilised. However, immobilisation onto solid supports greatly reduces the volumetric and specific activity of the biocatalysts.