A Simple and Effective Cleavable Linker for Chemical Proteomics Applications

The study of metabolically labeled or probe-modified proteins is an important area in chemical proteomics. Isolation and purification of the protein targets is a necessary step before MS identification. The biotin-streptavidin system is widely used in this process, but the harsh denaturing conditions also release natively biotinylated proteins and non-selectively bound proteins. A cleavable linker strategy is a promising approach for solving this problem. Though several cleavable linkers have been developed and tested, an efficient, easily synthesized, and inexpensive cleavable linker is a desirable addition to the proteomics toolbox. Here, we describe the chemical proteomics application of a vicinal diol cleavable linker. Through easy-to-handle chemistry we incorporate this linker into an activity-based probe and a biotin alkyne tag amenable for bioorthogonal ligation. With these reagents, background protein identifications are significantly reduced relative to standard on-bead digestion.The covalent modification of proteins by small molecules within a complex proteome is a major theme in chemical biology and proteomics. An effective method for the detection of posttranslational modifications of proteins is the metabolic incorporation of modified biomolecules such as tagged carbohydrates or lipids (1). Reversible interactions of enzyme inhibitors, natural products, or drugs can be detected when one appends photocrosslinking agents, thereby facilitating target discovery (2, 3). A particularly interesting example of protein labeling is activity-based protein profiling (ABPP)¹ (4, 5), which utilizes the intrinsic catalytic activity of a target enzyme for the covalent attachment of an affinity or visualization tag. ABPP makes use of small molecules (activity-based probes (ABPs)) that react with the active form of a specific enzyme or enzyme class by means of a “warhead,” which is often derived from a mechanism-based enzyme inhibitor (Fig. 1A). DCG-04, for example, is based on the naturally occurring inhibitor E-64 and targets the papain family of cysteine proteases via covalent attachment of the epoxysuccinate group to the active site cysteine (Fig. 1B) (6).Open in a separate windowFig. 1.The cleavable linker strategy in ABPP. A, the elements of an ABP. B, the example ABP DCG-04, an epoxysuccinate-containing probe for clan CA cysteine proteases. DCG-04 is based on the naturally occurring protease inhibitor E-64. C, schematic strategy of cleavable linker-mediated target identification. D, the cleavage mechanism of a vicinal diol.Bulky fluorophore or biotin tags on chemical probes might interfere with efficient protein binding. Moreover, they can negatively influence the cell permeability of probes, which therefore limits their applicability in in vitro experiments. Bioorthogonal chemistries, such as the Bertozzi-Staudinger ligation (7) and the 1,3-bipolar cycloaddition of an azide and an alkyne (click chemistry) (8), allow tandem labeling strategies in which a biotin or a fluorophore is attached to an enzyme probe complex in a separate step. Consequently, the probes themselves only carry azide or alkyne groups as “mini-tags.” Tandem labeling using bioorthogonal chemistry has now become a widely used strategy to label biomolecules in lysates and in live cells (9–11).An essential step in ABPP, as well as in other chemical proteomics approaches, is the elucidation of the tagged proteins. This usually involves a biotin-mediated enrichment step followed by mass-spectrometry-based identification. Although the streptavidin-biotin interaction allows efficient enrichment as a result of the strong binding affinity (K_d ∼ 10⁻¹⁵ m), it also has limitations. The quantitative elution of biotinylated proteins requires harsh conditions (12), which lead to contamination of the sample by endogenous biotinylated and non-specifically bound proteins. These other proteins will be identified together with the real protein targets. Given that subsequent target validation with secondary assays can be a costly and time-consuming process, a reduction in false positive identifications is highly desirable. For cleaner protein identification, cleavable linker strategies (13) that allow the selective release of target proteins have been developed (Fig. 1C). The commercially available disulfide linker can be cleaved under mild conditions, but it suffers from premature cleavage in reducing media such as the intracellular environment and reducing buffers used for click chemistry and in vitro reactions of cysteine proteases. Therefore, a variety of alternative linkers for proteomics applications have been reported, including a sterically hindered disulfide (14), diazobenzenes (15–19), hydrazones (20, 21), silanes (22), light sensitive linkers (23–25), tobacco etch virus protease sensitive linkers (26, 27), and a levulinoyl-based linker (28). The synthesis of some of these linkers is lengthy or difficult to scale up, which limits their general application in chemical proteomics.Ideally, a cleavable linker is stable under a wide variety of conditions, is efficiently and selectively cleaved, and can be synthesized in a low number of easy chemical transformations. We aimed to meet these requirements by using a vicinal diol as a cleavable linker system. When vicinal diols are treated with sodium periodate (NaIO₄), the carbon–carbon bond is cleaved (Fig. 1D). Periodate treatment of proteins can result in side-reactions, such as the cleavage of linked carbohydrates or the oxidation of N-terminal serine and threonine residues. However, these N-termini rarely occur in proteins and are therefore of minor concern. In general, the mild, neutral conditions of periodate cleavage are compatible with proteins. This has been illustrated in the past, for example, by its application in the detection of protein–protein interactions (29) and the creation of unliganded MHC class I molecules (30). In this article, we report the chemical proteomics application of diol cleavable linker probes. We show that the synthesis of the linker and its probe derivatives is straightforward, that the linker is compatible with tandem click labeling, that enrichment and release of probe targets is efficient, and that the identification of targets takes place with significantly lower background than in on-bead digestion protocols.     

Cryo‐Immunoelectron Microscopy of Adherent Cells Improved by the Use of Electrospun Cell Culture Substrates

Electrospun nanofibres are an excellent cell culture substrate, enabling the fast and non‐disruptive harvest and transfer of adherent cells for microscopical and biochemical analyses. Metabolic activity and cellular structures are maintained during the only half a minute‐long harvest and transfer process. We show here that such samples can be optimally processed by means of cryofixation combined either with freeze‐substitution, sample rehydration and cryosection‐immunolabelling or with freeze‐fracture replica‐immunolabelling. Moreover, electrospun fibre substrates are equally suitable for complementary approaches, such as biochemistry, fluorescence microscopy and cytochemistry.     

Task Dependency of Grip Stiffness—A Study of Human Grip Force and Grip Stiffness Dependency during Two Different Tasks with Same Grip Forces

It is widely known that the pinch-grip forces of the human hand are linearly related to the weight of the grasped object. Less is known about the relationship between grip force and grip stiffness. We set out to determine variations to these dependencies in different tasks with and without visual feedback. In two different settings, subjects were asked to (a) grasp and hold a stiffness-measuring manipulandum with a predefined grip force, differing from experiment to experiment, or (b) grasp and hold this manipulandum of which we varied the weight between trials in a more natural task. Both situations led to grip forces in comparable ranges. As the measured grip stiffness is the result of muscle and tendon properties, and since muscle/tendon stiffness increases more-or-less linearly as a function of muscle force, we found, as might be predicted, a linear relationship between grip force and grip stiffness. However, the measured stiffness ranges and the increase of stiffness with grip force varied significantly between the two tasks. Furthermore, we found a strong correlation between regression slope and mean stiffness for the force task which we ascribe to a force stiffness curve going through the origin. Based on a biomechanical model, we attributed the difference between both tasks to changes in wrist configuration, rather than to changes in cocontraction. In a new set of experiments where we prevent the wrist from moving by fixing it and resting it on a pedestal, we found subjects exhibiting similar stiffness/force characteristics in both tasks.     

A CNGB1 Frameshift Mutation in Papillon and Phalène Dogs with Progressive Retinal Atrophy

Progressive retinal degenerations are the most common causes of complete blindness both in human and in dogs. Canine progressive retinal atrophy (PRA) or degeneration resembles human retinitis pigmentosa (RP) and is characterized by a progressive loss of rod photoreceptor cells followed by a loss of cone function. The primary clinical signs are detected as vision impairment in a dim light. Although several genes have been associated with PRAs, there are still PRAs of unknown genetic cause in many breeds, including Papillons and Phalènes. We have performed a genome wide association and linkage studies in cohort of 6 affected Papillons and Phalènes and 14 healthy control dogs to map a novel PRA locus on canine chromosome 2, with a 1.9 Mb shared homozygous region in the affected dogs. Parallel exome sequencing of a trio identified an indel mutation, including a 1-bp deletion, followed by a 6-bp insertion in the CNGB1 gene. This mutation causes a frameshift and premature stop codon leading to probable nonsense mediated decay (NMD) of the CNGB1 mRNA. The mutation segregated with the disease and was confirmed in a larger cohort of 145 Papillons and Phalènes (P_Fisher = 1.4×10⁻⁸) with a carrier frequency of 17.2 %. This breed specific mutation was not present in 334 healthy dogs from 10 other breeds or 121 PRA affected dogs from 44 other breeds. CNGB1 is important for the photoreceptor cell function its defects have been previously associated with retinal degeneration in both human and mouse. Our study indicates that a frameshift mutation in CNGB1 is a cause of PRA in Papillons and Phalènes and establishes the breed as a large functional animal model for further characterization of retinal CNGB1 biology and possible retinal gene therapy trials. This study enables also the development of a genetic test for breeding purposes.     

Phylogenetic and population genetic analyses suggest a potential species boundary between Mountain (Gazella gazella) and Arabian Gazelles (G. arabica) in the Levant

Two cryptic lineages of ‘Mountain Gazelles’ have been reported based on molecular phylogenetic analyses using maternally inherited (mitochondrial) sequence markers, namely Gazella gazella in the Levant and G. arabica south of the Arava Valley into the Arabian Peninsula. Here, we provide a rigorous test for the existence of two distinct lineages based on bi-parentally inherited (nuclear microsatellite) markers. Our study confirms two genetically distinct clusters in the Levant and detected no gene-flow between them. Divergence time (inferred from a cytochrome b-based phylogeny) was approximately one MYA. Treating and breeding both lineages separately in future conservation and captive breeding programmes is highly recommended.     

Sex-Differences of Face Coding: Evidence from Larger Right Hemispheric M170 in Men and Dipole Source Modelling

The processing of faces relies on a specialized neural system comprising bilateral cortical structures with a dominance of the right hemisphere. However, due to inconsistencies of earlier findings as well as more recent results such functional lateralization has become a topic of discussion. In particular, studies employing behavioural tasks and electrophysiological methods indicate a dominance of the right hemisphere during face perception only in men whereas women exhibit symmetric and bilateral face processing. The aim of this study was to further investigate such sex differences in hemispheric processing of personally familiar and opposite-sex faces using whole-head magnetoencephalography (MEG). We found a right-lateralized M170-component in occipito-temporal sensor clusters in men as opposed to a bilateral response in women. Furthermore, the same pattern was obtained in performing dipole localization and determining dipole strength in the M170-timewindow. These results suggest asymmetric involvement of face-responsive neural structures in men and allow to ascribe this asymmetry to the fusiform gyrus. This specifies findings from previous investigations employing event-related potentials (ERP) and LORETA reconstruction methods yielding rather extended bilateral activations showing left asymmetry in women and right lateralization in men. We discuss our finding of an asymmetric fusiform activation pattern in men in terms of holistic face processing during face evaluation and sex differences with regard to visual strategies in general and interest for opposite faces in special. Taken together the pattern of hemispheric specialization observed here yields new insights into sex differences in face perception and entails further questions about interactions between biological sex, psychological gender and influences that might be stimulus-driven or task dependent.     

Ligand binding and dynamics of the monomeric epidermal growth factor receptor ectodomain

The ectodomain of the human epidermal growth factor receptor (hEGFR) controls input to several cell signalling networks via binding with extracellular growth factors. To gain insight into the dynamics and ligand binding of the ectodomain, the hEGFR monomer was subjected to molecular dynamics simulation. The monomer was found to be substantially more flexible than the ectodomain dimer studied previously. Simulations where the endogeneous ligand EGF binds to either Subdomain I or Subdomain III, or where hEGFR is unbound, show significant differences in dynamics. The molecular mechanics Poisson–Boltzmann surface area method has been used to derive relative free energies of ligand binding, and we find that the ligand is capable of binding either subdomain with a slight preference for III. Alanine‐scanning calculations for the effect of selected ligand mutants on binding reproduce the trends of affinity measurements. Taken together, these results emphasize the possible role of the ectodomain monomer in the initial step of ligand binding, and add details to the static picture obtained from crystal structures. Proteins 2013; 81:1931–1943. © 2013 The Authors. Proteins published by Wiley Periodicals, Inc.     

Immunohistochemical localization of caffeine in young Camellia sinensis (L.) O. Kuntze (tea) leaves

The anatomical localization of caffeine within young Camellia sinensis leaves was investigated using immunohistochemical methods and confocal scanning laser microscopy. Preliminary fixation experiments were conducted with young C. sinensis leaves to determine which fixation procedure retained caffeine the best as determined by high-performance liquid chromatography analysis. High pressure freezing, freeze substitution, and embedding in resin was deemed the best protocol as it retained most of the caffeine and allowed for the samples to be sectioned with ease. Immunohistochemical localization with primary anti-caffeine antibodies and conjugated secondary antibodies on leaf sections proved at the tissue level that caffeine was localized and accumulated within vascular bundles, mainly the precursor phloem. With the use of a pressure bomb, xylem sap was collected using a micro syringe. The xylem sap was analyzed by thin-layer chromatography and the presence of caffeine was determined. We hypothesize that caffeine is synthesized in the chloroplasts of photosynthetic cells and transported to vascular bundles where it acts as a chemical defense against various pathogens and predators. Complex formation of caffeine with chlorogenic acid is also discussed as this may also help explain caffeine’s localization.     

Camphor Pathway Redux: Functional Recombinant Expression of 2,5- and 3,6-Diketocamphane Monooxygenases of Pseudomonas putida ATCC 17453 with Their Cognate Flavin Reductase Catalyzing Baeyer-Villiger Reactions

Whereas the biochemical properties of the monooxygenase components that catalyze the oxidation of 2,5-diketocamphane and 3,6-diketocamphane (2,5-DKCMO and 3,6-DKCMO, respectively) in the initial catabolic steps of (+) and (−) isomeric forms of camphor (CAM) metabolism in Pseudomonas putida ATCC 17453 are relatively well characterized, the actual identity of the flavin reductase (Fred) component that provides the reduced flavin to the oxygenases has hitherto been ill defined. In this study, a 37-kDa Fred was purified from a camphor-induced culture of P. putida ATCC 17453 and this facilitated cloning and characterization of the requisite protein. The active Fred is a homodimer with a subunit molecular weight of 18,000 that uses NADH as an electron donor (K_m = 32 μM), and it catalyzes the reduction of flavin mononucleotide (FMN) (K_m = 3.6 μM; k_cat = 283 s⁻¹) in preference to flavin adenine dinucleotide (FAD) (K_m = 19 μM; k_cat = 128 s⁻¹). Sequence determination of ∼40 kb of the CAM degradation plasmid revealed the locations of two isofunctional 2,5-DKCMO genes (camE_25–1 for 2,5-DKCMO-1 and camE_25–2 for 2,5-DKCMO-2) as well as that of a 3,6-DKCMO-encoding gene (camE₃₆). In addition, by pulsed-field gel electrophoresis, the CAM plasmid was established to be linear and ∼533 kb in length. To enable functional assessment of the two-component monooxygenase system in Baeyer-Villiger oxidations, recombinant plasmids expressing Fred in tandem with the respective 2,5-DKCMO- and 3,6-DKCMO-encoding genes in Escherichia coli were constructed. Comparative substrate profiling of the isofunctional 2,5-DCKMOs did not yield obvious differences in Baeyer-Villiger biooxidations, but they are distinct from 3,6-DKCMO in the stereoselective oxygenations with various mono- and bicyclic ketone substrates.     

Invasion genetics of American cherry fruit fly in Europe and signals of hybridization with the European cherry fruit fly

The American cherry fruit fly is an invasive pest species in Europe, of serious concern in tart cherry production as well as for the potential to hybridize with the European cherry fruit fly, Rhagoletis cerasi L. (Diptera: Tephritidae), which might induce new pest dynamics. In the first European reports, the question arose whether only the eastern American cherry fruit fly, Rhagoletis cingulata (Loew) (Diptera: Tephritidae), is present, or also the closely related western American cherry fruit fly, Rhagoletis indifferens Curran. In this study, we investigate the species status of European populations by comparing these with populations of both American species from their native ranges, the invasion dynamics in German (first report in 1993) and Hungarian (first report in 2006) populations, and we test for signals of hybridization with the European cherry fruit fly. Although mtDNA sequence genealogy could not separate the two American species, cross‐species amplification of 14 microsatellite loci separated them with high probabilities (0.99–1.0) and provided evidence for R. cingulata in Europe. German and Hungarian R. cingulata populations differed significantly in microsatellite allele frequencies, mtDNA haplotype and wing pattern distributions, and both were genetically depauperate relative to North American populations. The diversity suggests independent founding events in Germany and Hungary. Within each country, R. cingulata displayed little or no structure in any trait, which agrees with rapid local range expansions. In cross‐species amplifications, signals of hybridization between R. cerasi and R. cingulata were found in 2% of R. cingulata individuals and in 3% of R. cerasi. All putative hybrids had R. cerasi mtDNA indicating that the original between‐species mating involved R. cerasi females and R. cingulata males.