Cleavable Crosslinkers as Tissue Fixation Reagents for Proteomic Analysis

Formaldehyde fixation is widely used for long‐term maintenance of tissue. However, due to formaldehyde‐induced crosslinks, fixed tissue proteins are difficult to extract, which hampers mass spectrometry (MS) proteomic analyses. Recent years have seen the use of different combinations of high temperature and solubilizing agents (usually derived from antigen retrieval techniques) to unravel formaldehyde‐fixed paraffin‐embedded tissue proteomes. However, to achieve protein extraction yields similar to those of fresh‐frozen tissue, high‐temperature heating is necessary. Such harsh extraction conditions can affect sensitive amino acids and post‐translational modifications, resulting in the loss of important information, while still not resulting in protein yields comparable to those of fresh‐frozen tissue. Herein, the objective is to evaluate cleavable protein crosslinkers as fixatives that allow tissue preservation and efficient protein extraction from fixed tissue for MS proteomics under mild conditions. With this goal in mind, disuccinimidyl tartrate (DST) and dithiobis(succinimidylpropionate) (DSP) are investigated as cleavable fixating reagents. These compounds crosslink proteins by reacting with amino groups, leading to amide bond formation, and can be cleaved with sodium metaperiodate (cis‐diols, DST) or reducing agents (disulfide bonds, DSP), respectively. Results show that cleavable protein crosslinking with DST and DSP allows tissue fixation with morphology preservation comparable to that of formaldehyde. In addition, cleavage of DSP improves protein recovery from fixed tissue by a factor of 18 and increases the number of identified proteins by approximately 20 % under mild extraction conditions compared with those of formaldehyde‐fixed paraffin‐embedded tissue. A major advantage of DSP is the introduction of well‐defined protein modifications that can be taken into account during database searching. In contrast to DSP fixation, DST fixation followed by cleavage with sodium metaperiodate, although effective, results in side reactions that prevent effective protein extraction and interfere with protein identification. Protein crosslinkers that can be cleaved under mild conditions and result in defined modifications, such as DSP, are thus viable alternatives to formaldehyde as tissue fixatives to facilitate protein analysis from paraffin‐embedded, fixed tissue.     

Targeting G Protein‐Coupled Receptors by Capture Compound Mass Spectrometry: A Case Study with Sertindole

Unbiased chemoproteomic profiling of small‐molecule interactions with endogenous proteins is important for drug discovery. For meaningful results, all protein classes have to be tractable, including G protein‐coupled receptors (GPCRs). These receptors are hardly tractable by affinity pulldown from lysates. We report a capture compound (CC)‐based strategy to target and identify GPCRs directly from living cells. We synthesized CCs with sertindole attached to the CC scaffold in different orientations to target the dopamine D2 receptor (DRD2) heterologously expressed in HEK 293 cells. The structure–activity relationship of sertindole for DRD2 binding was reflected in the activities of the sertindole CCs in radioligand displacement, cell‐based assays, and capture compound mass spectrometry (CCMS). The activity pattern was rationalized by molecular modelling. The most‐active CC showed activities very similar to that of unmodified sertindole. A concentration of DRD2 in living cells well below 100 fmol used as an experimental input was sufficient for unambiguous identification of captured DRD2 by mass spectrometry. Our new CCMS workflow broadens the arsenal of chemoproteomic technologies to close a critical gap for the comprehensive characterization of drug–protein interactions.     

Chemical Genetics Approach to Engineer Kinesins with Sensitivity towards a Small‐Molecule Inhibitor of Eg5

Due to their fast and often reversible mode of action, small molecules are ideally suited to dissect biological processes. Yet, the validity of small‐molecule studies is intimately tied to the specificity of the applied compounds, thus imposing a great challenge to screens for novel inhibitors. Here, we applied a chemical‐genetics approach to render kinesin motor proteins sensitive to inhibition by the well‐characterized small molecule S‐Trityl‐l ‐cysteine (STLC). STLC specifically inhibits the kinesin Eg5 through binding to a known allosteric site within the motor domain. Transfer of this allosteric binding site into the motor domain of the human kinesins Kif3A and Kif4A sensitizes them towards STLC. Single‐molecule microscopy analyses confirmed that STLC inhibits the movement of chimeric but not wild‐type Kif4A along microtubules. Thus, our proof‐of‐concept study revealed that this chemical‐genetic approach provides a powerful strategy to specifically inhibit kinesins in vitro for which small‐molecule inhibitors are not yet available.     

Design and Synthesis of Mechano‐Responsive Color‐Changing Thermoplastic Elastomer Based on Poly(n‐Butyl Acrylate)–Spiropyran‐Polystyrene Comb‐Structured Graft Copolymers

Colorimetric mechanophores like spiropyran (SP) represent an emerging type of interesting signal molecule that can be incorporated into polymers or other materials as a stress transducer. In this work, a new type of spiropyran‐containing inimer molecule MA‐SP‐Br are designed and synthesized, which is incorporated into polybutylacrylate (PBA) chains through reversible addition‐fragmentation chain transfer (RAFT) copolymerization with n‐butyl acrylate (BA). PBA‐SP‐Br is then used as a macro‐initiator to graft polystyrene (PS) side chains from the PBA backbone through atom transfer radical polymerization (ATRP) of styrene. The resulting comb‐structured graft copolymer PBA‐SP‐PS contains 0.15–0.34% SP and exhibits a characteristic feature of thermoplastic elastomers. Under uniaxial stretch, the materials possess an excellent mechano‐responsivity and change color at strains as low as about 14%.     

Determination of hemin-binding characteristics of proteins by a combinatorial peptide library approach

Studies of the binding of heme/hemin to proteins or peptides have recently intensified as it became evident that heme serves not only as a prosthetic group, but also as a regulator and effector molecule interacting with transmembrane and cytoplasmic proteins. The iron‐ion‐containing heme group can associate with these proteins in different ways, with the amino acids Cys, His, and Tyr allowing individual modes of binding. Strong coordinate‐covalent binding, such as in cytochrome c, is known, and reversible attachment is also discussed. Ligands for both types of binding have been reported independently, though sometimes with different affinities for similar sequences. We applied a combinatorial approach using the library (X)₄(C/H/Y)(X)₄ to characterize peptide ligands with considerable hemin binding capacities. Some of the library‐selected peptides were comparable in terms of hemin association independently of whether or not a cysteine residue was present in the sequence. Indeed, a preference for His‐based (≈39 %) and Tyr‐based (≈40 %) sequences over Cys‐based ones (≈21 %) was detected. The binding affinities for the library‐selected peptides, as determined by UV/Vis spectroscopy, were in the nanomolar range. Moreover, selected representatives efficiently competed for hemin binding with the human BK channel hSlo1, which is known to be regulated by heme through binding to its heme‐binding domain.     

Assessing Time‐Reversibility Under Minimal Assumptions

Abstract. This article considers a simple procedure for assessing whether a weakly dependent univariate stochastic process is time‐reversible. Our approach is based on a simple index of the deviation from zero of the median of the one‐dimensional marginal law of differenced data. An attractive feature of the method is that it requires no moment assumptions. Instead of relying on Gaussian asymptotic approximations, we consider using subsampling and resampling methods to construct confidence intervals for the time‐reversibility parameter, and show that such inference procedures are asymptotically valid under a mild mixing condition. The small‐sample properties of the proposed procedures are examined by means of Monte Carlo experiments and an application to real‐world data is also presented.     

Secretion-and-capture cell-surface display for selection of target-binding proteins

This report describes a new cell-surface display system, the Secretion and Capture Technology (SECANT?) platform, which relies on in vivo biotinylation of the protein of interest followed by its capture on the avidinated surface of the parent cell. Cell sorting techniques are then used to isolate clones that display target-binding protein. A distinguishing feature of this method is its ability to display complex proteins, such as full-length immunoglobulin G (IgG) antibodies, on living cells. In this proof-of-concept study, Saccharomyces cerevisiae cells that displayed Herceptin IgG were isolated from a 10,000-fold excess of cells that displayed a lysozyme-binding antibody.     

Sequence‐Specific Covalent Capture Coupled with High‐Contrast Nanopore Detection of a Disease‐Derived Nucleic Acid Sequence

Hybridization‐based methods for the detection of nucleic acid sequences are important in research and medicine. Short probes provide sequence specificity, but do not always provide a durable signal. Sequence‐specific covalent crosslink formation can anchor probes to target DNA and might also provide an additional layer of target selectivity. Here, we developed a new crosslinking reaction for the covalent capture of specific nucleic acid sequences. This process involved reaction of an abasic (Ap) site in a probe strand with an adenine residue in the target strand and was used for the detection of a disease‐relevant T→A mutation at position 1799 of the human BRAF kinase gene sequence. Ap‐containing probes were easily prepared and displayed excellent specificity for the mutant sequence under isothermal assay conditions. It was further shown that nanopore technology provides a high contrast—in essence, digital—signal that enables sensitive, single‐molecule sensing of the cross‐linked duplexes.     

Nonhomogeneous flow of micellar solutions: A kinetic—network theory approach

The rheological behavior of micellar solutions is analyzed under nonhomogeneous velocity and stress flow conditions. The framework is based on the extended irreversible thermodynamics and the transient network formulation coupled to the underlying kinetics embodying two relevant processes: formation of wormlike chains from a free micellar solution through a thermally activated process and their flow induced degradation. The second kinetic process consists in the formation of entanglements from the free wormlike chains and their flow‐induced breakage. These processes are modeled in a coupled kinetic scheme constituted by a set of reversible kinetic equations describing the evolution in average of the three microstates (free short rod‐like micelles, free wormlike chains, and entangled wormlike chains) that reflect the complexity of macromolecular interactions. The predictions of the shear stress and first normal stress difference as a function of shear‐rate under banded flow are in good agreement with experimental data. © 2018 American Institute of Chemical Engineers AIChE J, 64: 2277–2292, 2018     

N‐doped porous carbons for CO2 capture: Rational choice of N‐containing polymer with high phenyl density as precursor

N‐doped porous carbons (NPCs) are highly promising for CO₂ capture, but their preparation is severely hindered by two factors, namely, the high cost of N‐containing polymer precursors and the low yield of carbon products. Here we report for the first time the fabrication of NPCs through the rational choice of the polymer NUT‐4, with low cost and high phenyl density, as precursor. For the material NPC‐600 obtained from carbonization at 600°C, the yield is as high as 52.1%. The adsorption capacity of CO₂ on NPC‐600 reaches 6.9 mmol/g at 273 K and 1 bar, which is obviously higher than that on the benchmarks, including 13X zeolite (4.1 mmol/g) and activated carbon (2.8 mmol/g), as well as most reported carbon materials. Our results also demonstrate that the present NPCs can be completely regenerated under mild conditions. The abundant microporosity and “CO₂‐philic” (N‐doped) sites are responsible for the adsorption performance. © 2016 American Institute of Chemical Engineers AIChE J, 63: 1648–1658, 2017     

Self‐Detecting and Self‐Healing Reinforce Elastomer Doped with Aggregation‐Induced Emission Molecules

Most of elastomers for fabrication of comfortable epidermal devices and smart actuators produce responsive signals by the stimuli‐induced deformation. Herein, a dynamic visualization of external stimuli rather than the deformation through synthesis of a self‐healing poly(dimethylsiloxane) (PDMS)‐based elastomer doped with aggregation‐induced emission (AIE) molecules is reported. The self‐healing PDMS‐based elastomer is designed and synthesized through molecule integration of reversible multi‐strength H‐bonds and permanent covalent crosslink sites. The adjustment of the weight ratio of elastic cross‐linker offers tunable mechanical properties of the resultant elastomer. After doping such an elastomer with AIE molecules of 1,1,2,2‐tetrakis(4‐nitrophenyl)ethane, the elastomer composite displays strong on–off fluorescence depending upon mechanical damage and temperatures, which can be used to detect the breaking and self‐healing performances, as well as the temperature change. The strategy described here provides another way to develop smart polymeric elastomers for practical applications.     

Glycan‐Mediated,Ligand‐Controlled Click Chemistry for Drug‐Target Identification

Membrane‐bound proteins are important pharmaceutical drug targets, yet few strategies exist for the identification of small‐molecule‐targeted membrane proteins in live‐cell systems. By exploiting metabolic glycan engineering of cell membrane proteins, we have developed an in situ glycan‐mediated ligand‐controlled click (“GLiCo‐Click”) chemistry methodology that enables the attachment of small‐molecule chemical probes to their receptor protein through glycans on live cells. In addition to enabling receptor enrichment from cell lysates, this strategy can be used to demonstrate target receptor engagement and enables the molecular characterization of receptors.     

Azide: A Unique Dipole for Metal‐Free Bioorthogonal Ligations

Covalently bound azide on a (small) organic molecule or a (large) biomolecular structure has proven an important handle for bioconjugation. Azides are readily introduced, small, and stable, yet undergo smooth ligation with a range of reactive probes under mild conditions. In particular, the potential of azides to undergo metal‐free reactions with strained unsaturated systems has inspired the development of an increasing number of reactive probes, which are comprehensively summarized here. For each individual probe, the synthetic preparation is described, together with reaction kinetics and the full range of applications, from materials science to glycoprofiling. Finally, a qualitative and quantitative comparison of azido‐reactive probes is provided.     

A sultone‐based reversible dark red‐yellow conversion thermochromic colorant with adjustable switching temperature

A reversible, adjustable thermochromic colorant with bichromatic conversion from dark red (cool state) to yellow (heat state) was prepared using bromocresol purple (BCP) as colour former. The thermochromic behaviour of this colorant was investigated during the heating‐cooling cycle, including the colour depth, colour difference and switching temperature. The mechanism of thermochromic colorant switching between dark red and yellow originated from the transformation of the conjugated structures of BCP, which was confirmed by infrared spectrometry. The switching temperature can be flexibility adjusted between 13–46 °C by mixing solvents according to Schröder's equation, which was demonstrated by differential scanning calorimetry measurement. The thermochromic colorant presented good reversible thermochromic performance with stable changes in colour parameters for 50 heating‐cooling cycles. This sultone‐based reversible dark red‐yellow conversion thermochromic colorant is suitable for application as thermal‐indicating material under various conditions.     

Pull-Down of Metalloproteins in Their Native States by Using Desthiobiotin-Based Probes

One-third of all proteins are estimated to require metals for structural stability and/or catalytic activity. Desthiobiotin probes containing metal binding groups can be used to capture metalloproteins with exposed active-site metals under mild conditions so as to prevent changes in metallation state. The proof-of-concept was demonstrated with carbonic anhydrase (CA), an open active site, Zn²⁺-containing protein. CA was targeted by using sulfonamide derivatives. Linkers of various lengths and structures were screened to determine the optimal structure for capture of the native protein. The optimized probes could selectively pull down CA from red blood cell lysate and other protein mixtures. Pull-down of differently metallated CAs was also investigated.     

Copper(I)‐Catalyzed Coupling Cyclization of Methyl Perfluoroalk‐2‐ynoates with 2‐Aminobenzonitriles: Synthesis of 2‐Perfluoroalkylated Quinolines

An efficient route to 2‐perfluoroalkylated quinoline derivatives through the copper(I)‐mediated coupling–cyclization of 2‐aminobenzonitriles with methyl perfluoroalk‐2‐ynoates is described. Moderate to excellent yields have been achieved under mild conditions. The reaction mechanism is also discussed.     

Stabilized Performance of Al‐Decorated and Al/Mg Co‐Decorated Spray‐Dried CaO‐Based CO2 Sorbents

The sharp loss‐in‐capacity in CO₂ capture as a result of sintering is a major drawback for CaO‐based sorbents used in the calcium looping process. The decoration of inert supports effectively stabilizes the cyclic CO₂ capture performance of CaO‐based sorbents via sintering mitigation. A range of Al‐decorated and Al/Mg co‐decorated CaO‐based sorbents were synthesized via an easily scaled‐up spray‐drying route. The decoration of Al‐based and Al/Mg‐based supports efficiently enhanced the cyclic CO₂ capture capability of CaO‐based sorbents under severe testing conditions. The CO₂ capture capacity losses of Al‐decorated and Al/Mg co‐decorated CaO‐based sorbents were alleviated, representing more stable CO₂ capture performance. The stabilized CO₂ capture performance is mainly attributed to the formation of Ca₁₂Al₁₄O₃₃, MgAl₂O₄, and MgO that act as the skeleton structures to mitigate the sintering of CaCO₃ during carbonation/calcination cycles.     

An Oxidative Bioconjugation Strategy Targeted to a Genetically Encoded 5‐Hydroxytryptophan

Approaches that enable the chemoselective, covalent modification of proteins in a site‐specific manner have emerged as a powerful technology for a wide range of applications. The electron‐rich unnatural amino acid 5‐hydroxytryptophan was recently genetically encoded in both Escherichia coli and eukaryotes, thereby allowing its site‐specific incorporation into virtually any recombinant protein. Herein, we report the chemoselective conjugation of various aromatic amines to full‐length proteins under mild, oxidative conditions that target this site‐specifically incorporated 5‐hydroxytryptophan residue.     

Comprehensive Discovery of the Accessible Primary Amino Group-Containing Segments from Cell Surface Proteins by Fine-Tuning a High-Throughput Biotinylation Method

Cell surface proteins, including transmembrane and other surface-anchored proteins, play a key role in several critical cellular processes and have a strong diagnostic value. The development of quick and robust experimental methods remains vital for the accurate and comprehensive characterization of the cell surface subproteome of individual cells. Here we present a high-throughput technique which relies on the biotinylation of the accessible primary amino groups in the extracellular segments of the proteins, using HL60 as a model cell line. Several steps of the method have been thoroughly optimized to capture labeled surface proteins selectively and in larger quantities. These include the following: improving the efficiency of the cell surface biotinylation; reducing the endogen protease activity; applying an optimal amount of affinity column and elution steps for labeled peptide enrichment; and examining the effect of various solid-phase extraction methods, different HPLC gradients, and various tandem mass spectrometry settings. Using the optimized workflow, we identified at least 1700 surface-associated individual labeled peptides (~6000–7000 redundant peptides) from the model cell surface in a single nanoHPLC-MS/MS run. The presented method can provide a comprehensive and specific list of the cell surface available protein segments that could be potential targets in various bioinformatics and molecular biology research.