Enrichment of Specifically Labeled Proteins by an Immobilized Host Molecule

Chemical proteomics relies primarily on click‐chemistry‐based protein labeling and biotin‐streptavidin enrichment, but these techniques have inherent limitations. Enrichment of intracellular proteins using a totally synthetic host–guest complex is described, overcoming the problem associated with the classical approach. We achieve this by affinity‐based protein labeling with a target‐specific probe molecule conjugated to a high‐affinity guest (suberanilohydroxamic acid–ammonium‐adamantane; SAHA‐Ad) and then enriching the labeled species using a cucurbit[7]uril bead. This method shows high specificity for labeled molecules in a MDA‐MB‐231 breast cancer cell lysate. Moreover, this method shows promise for labeling proteins in live cells.     

Super‐Chelators for Advanced Protein Labeling in Living Cells

Live‐cell labeling, super‐resolution microscopy, single‐molecule applications, protein localization, or chemically induced assembly are emerging approaches, which require specific and very small interaction pairs. The minimal disturbance of protein function is essential to derive unbiased insights into cellular processes. Herein, we define a new class of hexavalent N‐nitrilotriacetic acid (hexaNTA) chelators, displaying the highest affinity and stability of all NTA‐based small interaction pairs described so far. Coupled to bright organic fluorophores with fine‐tuned photophysical properties, the super‐chelator probes were delivered into human cells by chemically gated nanopores. These super‐chelators permit kinetic profiling, multiplexed labeling of His₆‐ and His₁₂‐tagged proteins as well as single‐molecule‐based super‐resolution imaging.     

Rapid protein concentration, efficient fluorescence labeling and purification on a micro/nanofluidics chip

Fluorescence analysis has proved to be a powerful detection technique for achieving single molecule analysis. However, it usually requires the labeling of targets with bright fluorescent tags since most chemicals and biomolecules lack fluorescence. Conventional fluorescence labeling methods require a considerable quantity of biomolecule samples, long reaction times and extensive chromatographic purification procedures. Herein, a micro/nanofluidics device integrating a nanochannel in a microfluidics chip has been designed and fabricated, which achieves rapid protein concentration, fluorescence labeling, and efficient purification of product in a miniaturized and continuous manner. As a demonstration, labeling of the proteins bovine serum albumin (BSA) and IgG with fluorescein isothiocyanate (FITC) is presented. Compared to conventional methods, the present micro/nanofluidics device performs about 10(4)-10(6) times faster BSA labeling with 1.6 times higher yields due to the efficient nanoconfinement effect, improved mass, and heat transfer in the chip device. The results demonstrate that the present micro/nanofluidics device promises rapid and facile fluorescence labeling of small amount of reagents such as proteins, nucleic acids and other biomolecules with high efficiency.     

Quantification of intact covalently metal labeled proteins using ESI‐MS/MS

Mass spectrometric methods matured from the successful qualitative characterization of proteins in complex mixtures into methods for quantitative proteomics often based on chemical tags with stable isotope labeling. In the study presented here, we extended the application of lanthanide‐ion‐based tags from the quantification using inductively coupled plasma‐MS into the quantification of labeled intact proteins using electrospray ionization (ESI)‐MS and ESI‐MS/MS. We applied the metal chelate tag MeCAT‐iodoacetamide (IA) (1,4,7,10‐tetraazacyclododecane N,N′,N″,N″ ′‐tetra acetic acid with a IA reactive site). Labeled proteins were separated using C3‐reversed phase‐high‐performance liquid chromatography interfaced to ESI‐MS. We could prove that even large proteins were completely labeled at all available cysteine residues using MeCAT‐IA with only a small excess of reagent. Fragmentation of labeled proteins either using infrared multiphoton dissociation in Fourier transform ion cyclotron resonance‐MS or higher‐energy collision dissociation with an Orbitrap gave characteristic fragments. We used these fragments to quantify several intact proteins avoiding digestion. To demonstrate the applicability, human serum albumin was quantified in blood serum. The high‐performance liquid chromatography/ESI‐MS/MS quantification data were validated using inductively coupled plasma‐MS. Because the metal within the tag may be any of the lanthanides, multiplexing capabilities are inherent. Copyright © 2014 John Wiley & Sons, Ltd.     

Chemoselective and Site‐Selective Lysine‐Directed Lysine Modification Enables Single‐Site Labeling of Native Proteins

The necessity for precision labeling of proteins emerged during the efforts to understand and regulate their structure and function. It demands selective attachment of tags such as affinity probes, fluorophores, and potent cytotoxins. Here, we report a method that enables single‐site labeling of a high‐frequency Lys residue in the native proteins. At first, the enabling reagent forms stabilized imines with multiple solvent‐accessible Lys residues chemoselectively. These linchpins create the opportunity to regulate the position of a second Lys‐selective electrophile connected by a spacer. Consequently, it enables the irreversible single‐site labeling of a Lys residue independent of its place in the reactivity order. The user‐friendly protocol involves a series of steps to deconvolute and address chemoselectivity, site‐selectivity, and modularity. Also, it delivers ordered immobilization and analytically pure probe‐tagged proteins. Besides, the methodology provides access to antibody‐drug conjugate (ADC), which exhibits highly selective anti‐proliferative activity towards HER‐2 expressing SKBR‐3 breast cancer cells.     

Red‐Emitting Rhodamine Dyes for Fluorescence Microscopy and Nanoscopy

Fluorescent markers emitting in the red are extremely valuable in biological microscopy since they minimize cellular autofluorescence and increase flexibility in multicolor experiments. Novel rhodamine dyes excitable with 630 nm laser light and emitting at around 660 nm have been developed. The new rhodamines are very photostable and have high fluorescence quantum yields of up to 80 %, long excited state lifetimes of 3.4 ns, and comparatively low intersystem‐crossing rates. They perform very well both in conventional and in subdiffraction‐resolution microscopy such as STED (stimulated emission depletion) and GSDIM (ground‐state depletion with individual molecular return), as well as in single‐molecule‐based experiments such as fluorescence correlation spectroscopy (FCS). Syntheses of lipophilic and hydrophilic derivatives starting from the same chromophore‐containing scaffold are described. Introduction of two sulfo groups provides high solubility in water and a considerable rise in fluorescence quantum yield. The attachment of amino or thiol reactive groups allows the dyes to be used as fluorescent markers in biology. Dyes deuterated at certain positions have narrow and symmetrical molecular mass distribution patterns, and are proposed as new tags in MS or LC‐MS for identification and quantification of various substance classes (e.g., amines and thiols) in complex mixtures. High‐resolution GSDIM images and live‐cell STED‐FCS experiments on labeled microtubules and lipids prove the versatility of the novel probes for modern fluorescence microscopy and nanoscopy.     

Site‐Specific Labeling of Proteins with a Chemically Stable,High‐Affinity Tag for Protein Study

Site‐specific labeling of proteins with paramagnetic lanthanides offers unique opportunities by virtue of NMR spectroscopy in structural biology. In particular, these paramagnetic data, generated by the anisotropic paramagnetism including pseudocontact shifts (PCS), residual dipolar couplings (RDC), and paramagnetic relaxation enhancement (PRE), are highly valuable in structure determination and mobility studies of proteins and protein–ligand complexes. Herein, we present a new way to label proteins in a site‐specific manner with a high‐affinity and chemically stable tag, 4‐vinyl(pyridine‐2,6‐diyl)bismethylenenitrilo tetrakis(acetic acid) (4VPyMTA), through thiol alkylation. Its performance has been demonstrated in G47C and E64C mutants of human ubiquitin both in vitro and in a crowded environment. In comparison with the published tags, 4VPyMTA has several interesting features: 1) it has a very high binding affinity for lanthanides (higher than EDTA), 2) there is no heterogeneity in complexes with lanthanides, 3) the derivatized protein is stable and potentially applicable to the in situ analysis of proteins.     

SLAP: Small Labeling Pair for Single‐Molecule Super‐Resolution Imaging

Protein labeling with synthetic fluorescent probes is a key technology in chemical biology and biomedical research. A sensitive and efficient modular labeling approach (SLAP) was developed on the basis of a synthetic small‐molecule recognition unit (Ni‐trisNTA) and the genetically encoded minimal protein His_6‐10‐tag. High‐density protein tracing by SLAP was demonstrated. This technique allows super‐resolution fluorescence imaging and fulfills the necessary sampling criteria for single‐molecule localization‐based imaging techniques. It avoids masking by large probes, for example, antibodies, and supplies sensitive, precise, and robust size analysis of protein clusters (nanodomains).     

Discerning the Chemistry in Individual Organelles with Small‐Molecule Fluorescent Probes

Principle has it that even the most advanced super‐resolution microscope would be futile in providing biological insight into subcellular matrices without well‐designed fluorescent tags/probes. Developments in biology have increasingly been boosted by advances of chemistry, with one prominent example being small‐molecule fluorescent probes that not only allow cellular‐level imaging, but also subcellular imaging. A majority, if not all, of the chemical/biological events take place inside cellular organelles, and researchers have been shifting their attention towards these substructures with the help of fluorescence techniques. This Review summarizes the existing fluorescent probes that target chemical/biological events within a single organelle. More importantly, organelle‐anchoring strategies are described and emphasized to inspire the design of new generations of fluorescent probes, before concluding with future prospects on the possible further development of chemical biology.     

Point Decoration of Silicon Nanowires: An Approach Toward Single‐Molecule Electrical Detection

Probing interactions of biological systems at the molecular level is of great importance to fundamental biology, diagnosis, and drug discovery. A rational bioassay design of lithographically integrating individual point scattering sites into electrical circuits is capable of realizing real‐time, label‐free biodetection of influenza H1N1 viruses with single‐molecule sensitivity and high selectivity by using silicon nanowires as local reporters in combination with microfluidics. This nanocircuit‐based architecture is complementary to more conventional optical techniques, but has the advantages of no bleaching problems and no fluorescent labeling. These advantages offer a promising platform for exploring dynamics of stochastic processes in biological systems and gaining information from genomics to proteomics to improve accurate molecular and even point‐of‐care clinical diagnosis.     

Kinetic Assay of the Michael Addition‐Like Thiol–Ene Reaction and Insight into Protein Bioconjugation

The chemical modification of proteins is a valuable technique in understanding the functions, interactions, and dynamics of proteins. Reactivity and selectivity are key issues in current chemical modification of proteins. The Michael addition‐like thiol–ene reaction is a useful tool that can be used to tag proteins with high selectivity for the solvent‐exposed thiol groups of proteins. To obtain insight into the bioconjugation of proteins with this method, a kinetic analysis was performed. New vinyl‐substituted pyridine derivatives were designed and synthesized. The reactivity of these vinyl tags with L ‐cysteine was evaluated by UV absorption and high‐resolution NMR spectroscopy. The results show that protonation of pyridine plays a key role in the overall reaction rates. The kinetic parameters were assessed in protein modification. The different reactivities of these vinyl tags with solvent‐exposed cysteine is valuable information in the selective labeling of proteins with multiple functional groups.     

Site-specific protein double labeling by expressed protein ligation: applications to repeat proteins

In the last few years, the use of labeled proteins has significantly expanded in the life sciences. Now, labeled proteins are indispensable tools for a wide spectrum of biophysical and chemical biology applications. In particular, the quest for more sophisticated experimental setups requires the development of new synthetic methodology, especially for multiple site-specific labeling. In this paper, we describe a synthetic strategy based on expressed protein ligation to prepare proteins in high purity and homogeneity, in which two different molecular probes are incorporated specifically at any desired position. Proteins are sequentially labeled in solution, with the advantage that a large excess of probes is not required and the labeled fragments are not restricted to peptide synthesis length limitations. This strategy was applied to selectively label a repeat protein with a fluorophores pair in different positions along the protein sequence. The doubly labeled proteins were prepared at high purity and homogeneity, as required for single molecule FRET studies. Remarkably, this approach can be adapted to the introduction of more than two molecular probes.     

Rhodium(II) Metallopeptide Catalyst Design Enables Fine Control in Selective Functionalization of Natural SH3 Domains

Chemically modified proteins are increasingly important for use in fundamental biophysical studies, chemical biology, therapeutic protein development, and biomaterials. However, chemical methods typically produce heterogeneous labeling and cannot approach the exquisite selectivity of enzymatic reactions. While bioengineered methods are sometimes an option, selective reactions of natural proteins remain an unsolved problem. Here we show that rhodium(II) metallopeptides combine molecular recognition with promiscuous catalytic activity to allow covalent decoration of natural SH3 domains, depending on choice of catalyst but independent of the specific residue present. A metallopeptide catalyst succeeds in modifying a single SH3‐containing kinase at endogenous concentrations in prostate cancer (PC‐3) cell lysate.     

Targeting the Small GTPase Superfamily through Their Regulatory Proteins

The Ras superfamily of small GTPases are guanine‐nucleotide‐dependent switches essential for numerous cellular processes. Mutations or dysregulation of these proteins are associated with many diseases, but unsuccessful attempts to target the small GTPases directly have resulted in them being classed as “undruggable”. The GTP‐dependent signaling of these proteins is controlled by their regulators; guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), and in the Rho and Rab subfamilies, guanine nucleotide dissociation inhibitors (GDIs). This review covers the recent small molecule and biologics strategies to target the small GTPases through their regulators. It seeks to critically re‐evaluate recent chemical biology practice, such as the presence of PAINs motifs and the cell‐based readout using compounds that are weakly potent or of unknown specificity. It highlights the vast scope of potential approaches for targeting the small GTPases in the future through their regulatory proteins.     

Ultrasensitive Detection and Binding Mechanism of Cocaine in an Aptamer‐based Single‐molecule Device

Aptamer serves as a potential candidate for the micro‐detection of cocaine due to its high specificity, high affinity and good stability. Although cocaine aptasensors have been extensively studied, the binding mechanism of cocaine‐aptamer interactions is still unknown, which limits the structural refinement in the design of an aptamer to improve the performance of cocaine aptasensors. Herein, we report a label‐free, ultrasensitive detection of single‐molecule cocaine‐aptamer interaction by using an electrical nanocircuit based on graphene‐molecule‐ graphene single‐molecule junctions (GMG‐SMJs). Real‐time recordings of cocaine‐aptamer interactions have exhibited distinct current oscillations before and after cocaine treatment, revealing the dynamic mechanism of the conformational changes of aptamer upon binding with cocaine. Further concentration‐dependent experiments have proved that these devices can act as a single‐molecule biosensor with at least a limit of detection as low as 1 nmol?L^–1. The method demonstrated in this work provides a novel strategy for shedding light on the interaction mechanism of biomolecules as well as constructing new types of aptasensors toward practical applications.     

Kinetically Tunable Photostability of Fluorogen‐Activating Peptide–Fluorogen Complexes

Ease of genetic encoding, labeling specificity, and high photostability are the most sought after qualities in a fluorophore for biological detection. Furthermore, many applications can gain from the fluorogenic nature of fluoromodules and the ability to turn on the same fluoromodules multiple times. Fluorogen‐activating peptides (FAPs) bind noncovalently to their cognate fluorogens and exhibit enhanced photostability. Herein, the photostabilities of malachite green (MG)‐binding and thiazole‐orange‐binding FAPs are compared under limiting‐ and excess‐fluorogen conditions to establish distinct mechanisms for photostability that correspond to the dissociation rate of the FAP–fluorogen complex. FAPs with slow dissociation show evidence of dye encapsulation and protection from photo or environmental degradation and single‐step bleaching at the single molecule level, whereas those with rapid dissociation show repeated cycles of binding and enhanced photostability by exchange of bleached fluorogen with a new dye. A combination of generalizable selection pressure based on bleaching, flow cytometry, and site‐specific amino acid mutagenesis is used to obtain a modified FAP with enhanced photostability, due to rapid dissociation of the MG fluorogen. These studies shed light on the basic mechanisms by which noncovalent association can effect photostable labeling, and demonstrate novel reagents for photostable and intermittent labeling of biological targets.     

Label‐Free Measurements of the Diffusivity of Molecules in Lipid Membranes

An important and characteristic property of a cell membrane is the lateral mobility of protein molecules in the lipid bilayer. This has conventionally been measured by labeling the molecules with fluorescent markers and monitoring their mobility by different fluorescence‐based techniques. However, adding the label to the studied molecule may affect the system, so it is an assumption in almost all experiments that the measured mobility of the biomolecule with its label is the same as that of the unlabeled molecule. However, this assumption is rarely tested due to a lack of suitable methods. In this work, a new technique to perform label‐free diffusivity measurements is developed and used to measure the effect of the label for two common protein–lipid systems: 1) streptavidin (SA) coupled to a supported lipid bilayer (SLB) through biotinylated lipids and 2) the extracellular part of the T‐cell adhesion protein CD2, coupled to an SLB through histidine tags to nickel‐chelating lipids. A measurable (≈12 %) decrease in diffusivity is found for both labeled proteins, even though the molecular mass of the label is almost 100 times smaller than those of the proteins (≈50 kDa). The results illustrate the importance of being able to study different biophysical properties of cell membranes and their mimics without relying on fluorescent labels, especially if fluorescent labeling is difficult or is expected to affect the nature of the intermolecular interactions being studied.     

Advances in Optical Single‐Molecule Detection: En Route to Supersensitive Bioaffinity Assays

The ability to detect low concentrations of analytes and in particular low‐abundance biomarkers is of fundamental importance, e.g., for early‐stage disease diagnosis. The prospect of reaching the ultimate limit of detection has driven the development of single‐molecule bioaffinity assays. While many review articles have highlighted the potentials of single‐molecule technologies for analytical and diagnostic applications, these technologies are not as widespread in real‐world applications as one should expect. This Review provides a theoretical background on single‐molecule—or better digital—assays to critically assess their potential compared to traditional analog assays. Selected examples from the literature include bioaffinity assays for the detection of biomolecules such as proteins, nucleic acids, and viruses. The structure of the Review highlights the versatility of optical single‐molecule labeling techniques, including enzymatic amplification, molecular labels, and innovative nanomaterials.     

Bioinspired Nitroalkylation for Selective Protein Modification and Peptide Stapling

Nitroalkanes react specifically with aldehydes, providing rapid, stable, and chemoselective protein bioconjugation. These nitroalkylated proteins mimic key post‐translational modifications (PTMs) of proteins and can be used to understand the role of these PTMs in cellular processes. Demonstrated here is the substrate scope of this bioconjugation by attaching a variety of tags, such as NMR tags, fluorescent tags, affinity tags, and alkyne tags, to proteins. The structure and enzymatic activity of modified proteins remain conserved after labeling. Notably, the nitroalkane group leads to easy characterization of proteins by mass spectrometry because of its distinct fingerprint pattern. Importantly, the nitro‐alkylated peptides provide a new handle for site‐selective fluorination of peptides, thus installing a specific probe to study peptide–protein interactions by ¹⁹F NMR spectroscopy. Furthermore, nitroalkane reagents can be used for the late‐stage diversification of peptides and for the synthesis of peptide staples.     

Protein Organic Chemistry and Applications for Labeling and Engineering in Live‐Cell Systems

The modification of proteins with synthetic probes is a powerful means of elucidating and engineering the functions of proteins both in vitro and in live cells or in vivo. Herein we review recent progress in chemistry‐based protein modification methods and their application in protein engineering, with particular emphasis on the following four strategies: 1) the bioconjugation reactions of amino acids on the surfaces of natural proteins, mainly applied in test‐tube settings; 2) the bioorthogonal reactions of proteins with non‐natural functional groups; 3) the coupling of recognition and reactive sites using an enzyme or short peptide tag–probe pair for labeling natural amino acids; and 4) ligand‐directed labeling chemistries for the selective labeling of endogenous proteins in living systems. Overall, these techniques represent a useful set of tools for application in chemical biology, with the methods 2–4 in particular being applicable to crude (living) habitats. Although still in its infancy, the use of organic chemistry for the manipulation of endogenous proteins, with subsequent applications in living systems, represents a worthy challenge for many chemists.