Chemical-Mediated Digestion: An Alternative Realm for Middle-down Proteomics?

Protein digestion in mass spectrometry (MS)-based bottom-up proteomics targets mainly lysine and arginine residues, yielding primarily 0.6-3 kDa peptides for the proteomes of organisms of all major kingdoms. Recent advances in MS technology enable analysis of complex mixtures of increasingly longer (>3 kDa) peptides in a high-throughput manner supporting the development of a middle-down proteomics (MDP) approach. Generating longer peptides is a paramount step in launching an MDP pipeline, but the quest for the selection of a cleaving agent that would provide the desired 3-15 kDa peptides remains open. Recent bioinformatics studies have shown that cleavage at the rarely occurring amino acid residues such as methionine (Met), tryptophan (Trp), or cysteine (Cys) would be suitable for MDP approach. Interestingly, chemical-mediated proteolytic cleavages uniquely allow targeting these rare amino acids, for which no specific proteolytic enzymes are known. Herein, as potential candidates for MDP-grade proteolysis, we have investigated the performance of chemical agents previously reported to target primarily Met, Trp, and Cys residues: CNBr, BNPS-Skatole (3-bromo-3-methyl-2-(2-nitrophenyl)sulfanylindole), and NTCB (2-nitro-5-thiobenzoic acid), respectively. Figures of merit such as digestion reproducibility, peptide size distribution, and occurrence of side reactions are discussed. The NTCB-based MDP workflow has demonstrated particularly attractive performance, and NTCB is put forward here as a potential cleaving agent for further MDP development.

Specification of haematopoietic stem cell fate via modulation of mitochondrial activity.

Haematopoietic stem cells (HSCs) differ from their committed progeny by relying primarily on anaerobic glycolysis rather than mitochondrial oxidative phosphorylation for energy production. However, whether this change in the metabolic program is the cause or the consequence of the unique function of HSCs remains unknown. Here we show that enforced modulation of energy metabolism impacts HSC self-renewal. Lowering the mitochondrial activity of HSCs by chemically uncoupling the electron transport chain drives self-renewal under culture conditions that normally induce rapid differentiation. We demonstrate that this metabolic specification of HSC fate occurs through the reversible decrease of mitochondrial mass by autophagy. Our data thus reveal a causal relationship between mitochondrial metabolism and fate choice of HSCs and also provide a valuable tool to expand HSCs outside of their native bone marrow niches.

A biocompatible in vivo ligation reaction and its application for noninvasive bioluminescent imaging of protease activity in living mice.

The discovery of biocompatible reactions had a tremendous impact on chemical biology, allowing the study of numerous biological processes directly in complex systems. However, despite the fact that multiple biocompatible reactions have been developed in the past decade, very few work well in living mice. Here we report that D-cysteine and 2-cyanobenzothiazoles can selectively react with each other in vivo to generate a luciferin substrate for firefly luciferase. The success of this "split luciferin" ligation reaction has important implications for both in vivo imaging and biocompatible labeling strategies. First, the production of a luciferin substrate can be visualized in a live mouse by bioluminescence imaging (BLI) and furthermore allows interrogation of targeted tissues using a "caged" luciferin approach. We therefore applied this reaction to the real-time noninvasive imaging of apoptosis associated with caspase 3/7. Caspase-dependent release of free D-cysteine from the caspase 3/7 peptide substrate Asp-Glu-Val-Asp-D-Cys (DEVD-(D-Cys)) allowed selective reaction with 6-amino-2-cyanobenzothiazole (NH(2)-CBT) in vivo to form 6-amino-D-luciferin with subsequent light emission from luciferase. Importantly, this strategy was found to be superior to the commercially available DEVD-aminoluciferin substrate for imaging of caspase 3/7 activity. Moreover, the split luciferin approach enables the modular construction of bioluminogenic sensors, where either or both reaction partners could be caged to report on multiple biological events. Lastly, the luciferin ligation reaction is 3 orders of magnitude faster than Staudinger ligation, suggesting further applications for both bioluminescence and specific molecular targeting in vivo.

Real-time noninvasive imaging of fatty acid uptake in vivo.

Detection and quantification of fatty acid fluxes in animal model systems following physiological, pathological, or pharmacological challenges is key to our understanding of complex metabolic networks as these macronutrients also activate transcription factors and modulate signaling cascades including insulin sensitivity. To enable noninvasive, real-time, spatiotemporal quantitative imaging of fatty acid fluxes in animals, we created a bioactivatable molecular imaging probe based on long-chain fatty acids conjugated to a reporter molecule (luciferin). We show that this probe faithfully recapitulates cellular fatty acid uptake and can be used in animal systems as a valuable tool to localize and quantitate in real time lipid fluxes such as intestinal fatty acid absorption and brown adipose tissue activation. This imaging approach should further our understanding of basic metabolic processes and pathological alterations in multiple disease models.