Evaluation of sulfatase-directed quinone methide traps for proteomics.

Sulfatases hydrolytically cleave sulfate esters through a unique catalytic aldehyde, which is introduced by a posttranslational oxidation. To profile active sulfatases in health and disease, activity-based proteomic tools are needed. Herein, quinone methide (QM) traps directed against sulfatases are evaluated as activity-based proteomic probes (ABPPs). Starting from a p-fluoromethylphenyl sulfate scaffold, enzymatically generated QM-traps can inactivate bacterial aryl sulfatases from Pseudomonas aeruginosa and Klebsiella pneumoniae, and human steroid sulfatase. However, multiple enzyme-generated QMs form, diffuse, and non-specifically label purified enzyme. In complex proteomes, QM labeling is sulfatase-dependent but also non-specific. Thus, fluoromethylphenyl sulfates are poor ABPPs for sulfatases.

Labeling and enrichment of Arabidopsis thaliana matrix metalloproteases using an active-site directed, marimastat-based photoreactive probe.

Matrix metalloproteases (MMPs) are secreted or membrane-bound zinc-containing proteases that play diverse roles in development and immunity in plants and in tissue remodeling in animals. We developed a photoreactive probe based on the MMP inhibitor marimastat, conjugated to a 4-azidotetrafluorobenzoyl moiety as photoreactive group and biotin as detection or sorting function. The probe labels At2-MMP, At4-MMP, At5-MMP, and likely other plant MMPs in leaf extracts, as shown by transient At-MMP expression in Nicotiana benthamiana, protein blot, and LC-MS/MS analysis. This MMP probe is a valuable tool to study the post-translational status of MMPs during plant immunity and other MMP-regulated processes.

Molecular tools for metalloprotease sub-proteome generation.

Molecular systems biology, the highly challenging post-genomic research area has many different facets like transcriptomics, proteomics, metabolomics, interactomics, modelling of cell cycles, etc. Among them, functional proteomics and interactomics represent exciting fields of research with high relevance towards biochemistry, medicinal chemistry, therapy, biotechnology and bioinformatics. The number of different proteins expressed by a cell under a set of certain conditions and the high dynamic range of these proteins together with different activation states require methods for sub-proteome generation on a mechanistic basis to reduce the amount of data. This can be achieved by application of tailor-made molecular tools that are based on inhibitors or, more generally, on protein ligands. Immobilised protein ligands proved to be suitable for the generation of sub-proteomes by affinity chromatography or by fishing using magnetic beads. Metalloproteases share a catalytically active metal ion in the active site. They can for example be addressed by hydroxamate type inhibitors like marimastat which are suitable for targeting active metalloproteases on a mechanistic basis aiming at the generation of an activity- and affinity-based sub-proteome. For such purposes, modified hydroxamate type inhibitors can be attached to a solid surface, e.g., chromatography material, magnetic beads, or a surface plasmon resonance sensor chip. The latter technique is a valuable tool for the optimisation of binding and elution conditions of biomolecules in affinity chromatography or on experiments using magnetic beads. Preliminary results are reported on the application of these probes in fishing experiments using magnetic beads.