Photocleavage of the Polypeptide Backbone by 2-Nitrophenylalanine

SummaryPhotocleavage of the polypeptide backbone is potentially a powerful and general method to activate or deactivate functional peptides and proteins with high spatial and temporal resolution. Here we show that 2-nitrophenylalanine is able to photochemically cleave the polypeptide backbone by an unusual cinnoline-forming reaction. This unnatural amino acid was genetically encoded in E. coli, and protein containing 2-nitrophenylalanine was expressed and site-specifically photocleaved

Proteasome inhibition for treatment of leishmaniasis, Chagas disease and sleeping sickness

Chagas disease, leishmaniasis and sleeping sickness affect 20 million people worldwide and lead to more than 50,000 deaths annually. The diseases are caused by infection with the kinetoplastid parasites Trypanosoma cruzi, Leishmania spp. and Trypanosoma brucei spp., respectively. These parasites have similar biology and genomic sequence, suggesting that all three diseases could be cured with drugs that modulate the activity of a conserved parasite target. However, no such molecular targets or broad spectrum drugs have been identified to date. Here we describe a selective inhibitor of the kinetoplastid proteasome (GNF6702) with unprecedented in vivo efficacy, which cleared parasites from mice in all three models of infection. GNF6702 inhibits the kinetoplastid proteasome through a non-competitive mechanism, does not inhibit the mammalian proteasome or growth of mammalian cells, and is well-tolerated in mice. Our data provide genetic and chemical validation of the parasite proteasome as a promising therapeutic target for treatment of kinetoplastid infections, and underscore the possibility of developing a single class of drugs for these neglected diseases

Exploring the phosphoproteome with mass spectrometry.

Protein phosphorylation is a reversible post-translational modification crucial in the control of numerous regulatory pathways. Understanding the highly interconnected nature of such networks requires new broader-scale analysis techniques. This report summarizes recent advances in the use of mass spectrometry to assess phosphorylation events in ever more complex systems

Sub-Zero Temperature Chromatography for Reduced Back-Exchange and Improved Dynamic Range in Amide Hydrogen Deuterium Exchange Mass Spectrometry

Amide hydrogen/deuterium exchange is a commonly used technique for studying the dynamics of proteins and their interactions with other proteins or ligands. When coupled with liquid chromatography and mass spectrometry, hydrogen/deuterium exchange provides several unique advantages over other structural characterization techniques including very high sensitivity, the ability to analyze proteins in complex environments, and a large mass range. A fundamental limitation of the technique arises from the loss of deuterium label (back-exchange) during the course of the analysis. A method to limit loss of label during the separation stage of the analysis using sub-zero temperature reversed-phase chromatography is presented. The approach is facilitated by the use of buffer modifiers that prevent freezing. We evaluated ethylene glycol, dimethyl formamide, formamide, and methanol for their freezing point suppression capabilities, effects on peptide retention, and their compatibilities with electrospray ionization. Ethylene glycol was used extensively because of its good electrospray ionization compatibility; however formamide has potential to be a superior modifier if detrimental effects on ionization can be overcome. It is demonstrated using suitable buffer modifiers that separations can be performed at temperatures as low as -30°C with negligible loss of deuterium label, even during long chromatographic separations. The reduction in back-exchange is shown to increase the dynamic range of HDX MS in terms of mixture complexity, and the magnitude with which changes in deuteration level can be quantified

Enrichment and analysis of peptide subsets using fluorous affinity tags and mass spectrometry.

Although mass spectrometry has become a powerful tool for the functional analysis of biological systems, complete proteome characterization cannot yet be achieved. Instead, the sheer complexity of living organisms demands fractionation of cellular extracts to enable more targeted analyses. Here, we introduce the concept of "fluorous proteomics," whereby specific peptide subsets from samples of biological origin are tagged with perfluorinated moieties and subsequently enriched by solid-phase extraction over a fluorous-functionalized stationary phase. This approach is extremely selective, yet can readily be tailored to enrich different subsets of peptides. Additionally, this methodology overcomes many of the limitations of traditional bioaffinity-based enrichment strategies, while enabling new affinity enrichment schemes impossible to implement with bioaffinity reagents. The potential of this methodology is demonstrated by the facile enrichment of peptides bearing particular side-chain functionalities or post-translational modifications from tryptic digests of individual proteins as well as whole cell lysates

Interlaboratory Comparison of Hydrogen-Deuterium Exchange Mass Spectrometry Measurements of the Fab fragment of NISTmAb

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is an established, powerful tool for investigating protein-ligand interactions, protein folding, and protein dynamics. However, HDX-MS is still an emergent tool for quality control of biopharmaceuticals and for establishing dynamic similarity between a biosimilar and an innovator therapeutic. Because industry will conduct quality control and similarity measurements over a product lifetime and in multiple locations, an understanding of HDX-MS reproducibility is critical. To determine the reproducibility of continuous-labeling, bottom-up HDX-MS measurements, the present interlaboratory comparison project evaluated deuterium uptake data from the Fab fragment of NISTmAb reference material (PDB: 5K8A) from fifteen laboratories. Laboratories reported ≈ 89,800 centroid measurements for 430 proteolytic peptide sequences of the Fab fragment (≈ 78,900 centroids), giving ≈ 100 % coverage, and ≈ 10,900 centroid measurements for 77 peptide sequences of the Fc fragment. Nearly half of peptide sequences are unique to the reporting laboratory, and only two sequences are reported by all laboratories. The majority of the laboratories (87 %) exhibited centroid mass laboratory repeatability precisions of 〈 sLab 〉 ≤ (0.15 ± 0.01) Da (1σx ̅ ), and all laboratories achieved 〈 sLab 〉 ≤ 0.4 Da. For immersions of protein at THDX = (3.6 to 25) oC and for D2O exchange times of tHDX = (30 s to 4 h) the reproducibility of back-exchange corrected, deuterium uptake measurements for the 15 laboratories is σreproducibility15 Labs ( tHDX ) = (9.0 ± 0.9) % (1σ). A 9 laboratory cohort that immersed samples at THDX = 25 oC exhibited reproducibility of σreproducibility25C cohort ( tHDX ) = (6.5 ± 0.6) % for back-exchange corrected, deuterium uptake measurements