High dynamic range proteome imaging with the structured illumination gel imager

A current challenge for proteomics is detecting proteins over the large concentration ranges found in complex biological samples such as whole‐cell extracts. Currently, no unbiased, whole‐proteome analysis scheme is capable of detecting the full range of cellular proteins. This is due in part to the limited dynamic range of the detectors used to sense proteins or peptides. We present a new technology, structured illumination (SI) gel imager, which detects fluorescently labeled proteins in electrophoretic gels over a 1 000 000‐fold concentration range. SI uses computer‐generated masks to attenuate the illumination of highly abundant proteins, allowing for long exposures of low‐abundance proteins, thus avoiding detector saturation. A series of progressively masked gel images are assembled into a single, very high dynamic range image. We demonstrate that the SI imager can detect proteins over a concentration range of approximately 1 000 000‐fold, making it a useful tool for comprehensive, unbiased proteome‐wide surveys.     

Tetrasubstituted pyrazinones derived from the reaction of praziquantel with N-bromosuccinimide

When praziquantel was exposed to N-bromosuccinimide in the presence of ethanol, a tricyclic 3-bromo-1-ethoxy pyrazinone was formed. From this and the analogous 1,3-dibromopyrazinone, a small library of 3-alkylamino-1-ethoxy, 1,3-dialkoxy, 3-alkoxy-1-bromo, and 3-alkylamino-1-bromo substituted pyrazinones were synthesized in high yields.     

Synthetic studies directed toward the AB decalin common to HMP-Y1 and hibarimicinone

Efforts toward the synthesis of the decalin ring system common to the hibarimicin shunt metabolite HMP-Y1 and parent aglycone hibarimicinone are reported herein. An intramolecular Diels–Alder cyclization rapidly generated the decalin framework. Two approaches toward completion of the AB decalin were vetted. Incorporation of a phenylsulfonyl leaving group β- to both a ketone and a γ-lactone followed by base-induced elimination of sulfinate led to the undesired α,β-unsaturated lactone. Methanolysis of the γ-lactone followed by elimination produced the unexpected bridged cyclic ether by way of an intramolecular oxy-Michael addition of the endo oriented C13 alcohol.     

An appraisal of the Suzuki cross-coupling reaction for the synthesis of novel fluorescent coumarin derivatives

We report the chemical design and development of 3-aryl-substituted 7-alkoxy-4-methylcoumarins with enhanced fluorogenic properties. The 3-aryl substituents are installed via an optimized Suzuki–Miyaura cross-coupling (SMC) reaction between a 7-alkoxy-3-bromo-4-methylcoumarin and aryl boronic MIDA esters using Pd(OAc)₂/XPhos in a catalytic system with K₂CO₃ in aqueous THF. Under these conditions, an exocyclic ester functionality is found to be unaffected. Subsequent saponification revealed a carboxylic acid functionality that is suitable for conjugation reactions. Evaluation of their fluorescence properties indicated that the installed 3-heteroaryl substituent, particularly benzofuran-2-yl, resulted in a significant red shift of both the excitation and emission wavelengths.     

Screen-printed electrode-based electrochemical detector coupled with in-situ ionic-liquid-assisted dispersive liquid–liquid microextraction for determination of 2,4,6-trinitrotoluene

A novel method is reported, whereby screen-printed electrodes (SPELs) are combined with dispersive liquid–liquid microextraction. In-situ ionic liquid (IL) formation was used as an extractant phase in the microextraction technique and proved to be a simple, fast and inexpensive analytical method. This approach uses miniaturized systems both in sample preparation and in the detection stage, helping to develop environmentally friendly analytical methods and portable devices to enable rapid and onsite measurement. The microextraction method is based on a simple metathesis reaction, in which a water-immiscible IL (1-hexyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide, [Hmim][NTf₂]) is formed from a water-miscible IL (1-hexyl-3-methylimidazolium chloride, [Hmim][Cl]) and an ion-exchange reagent (lithium bis[(trifluoromethyl)sulfonyl]imide, LiNTf₂) in sample solutions. The explosive 2,4,6-trinitrotoluene (TNT) was used as a model analyte to develop the method. The electrochemical behavior of TNT in [Hmim][NTf₂] has been studied in SPELs. The extraction method was first optimized by use of a two-step multivariate optimization strategy, using Plackett–Burman and central composite designs. The method was then evaluated under optimum conditions and a good level of linearity was obtained, with a correlation coefficient of 0.9990. Limits of detection and quantification were 7 μg L^?1 and 9 μg L^?1, respectively. The repeatability of the proposed method was evaluated at two different spiking levels (20 and 50 μg L^?1), and coefficients of variation of 7 % and 5 % (n?=?5) were obtained. Tap water and industrial wastewater were selected as real-world water samples to assess the applicability of the method.

Application of capillary electrophoresis for the early diagnosis of cancer

Early diagnosis is the key to the effective treatment of cancer. The detection of cancer biomarkers plays a critical role not only in cancer early diagnosis, but also in classification and staging tumor progression, or assessment prognosis and treatment response. Currently, various molecular diagnostic techniques have been developed for cancer biomarker studies, with many of the more effective approaches requiring a separation step before detection. Capillary electrophoresis (CE) can perform rapid and efficient separation with small samples, which is well-suited for analysis of both small- and macro- molecule biomarkers in complex samples. CE has different separation modes and can couple to different detectors into a variety of platforms, such as conducting studies on DNA/ RNA point mutation, protein misexpression, and metabolite abnormality. Similarly, microchip capillary electrophoresis (MCE) appears as a very important biomarker screening platform with the merits of high throughput, integration, and miniaturization, which makes it a promising clinical tool. By hyphenated different detectors, or integrated with immunoassay, PCR/LDR and related technologies, MCE can be constructed into diverse platforms used in genomics, proteomics, and metabolomics study for biomarkers discovery. The multiplex biomarker screening approach via CE- or MCE-based platforms is becoming a trend. This paper focuses on studies of cancer biomarkers via CE/MCE platforms, based on the studies published over the past 3 years. Some recent CE applications in the field of cancer study, such as cancer theranostics, are introduced.     

A Chemically Fuelled Molecular Automaton Displaying Programmed Migration of Zn2+ Between Alternative Binding Sites

A molecular system comprising a cationic zinc complex and an amino acid-derived ambident ligand having phosphate and carboxylate binding sites undergoes a series of rearrangements in which the metal cation migrates autonomously from one site to another. The location of the metal is identified by the circular dichroism spectrum of a ligated bis(2-quinolylmethyl)-(2-pyridylmethyl)amine (BQPA) chromophore, which takes a characteristic shape at each binding site. Migration is fuelled by the decomposition of trichloroacetic acid to CO₂ and CHCl₃, which progressively neutralises the acidity of the system as a function of time, revealing in sequence binding sites of increasing basicity. The migration rate responds to control by variation of the temperature, water content and triethylamine concentration, while an excess of fuel controls the duration of an induction period before the migration event.     

Sulfur and Azobenzenes,a Profitable Liaison: Straightforward Synthesis of Photoswitchable Thioglycosides with Tunable Properties

Azobenzene photoswitches are valuable tools for controlling properties of molecular systems with light. We have been investigating azobenzene glycoconjugates to probe carbohydrate-protein interactions and to design glycoazobenzene macrocycles with chiroptical and physicochemical properties modulated by light irradiation. To date, direct conjugation of glycosides to azobenzenes was performed by reactions providing target compounds in limited yields. We therefore sought a more effective and reliable coupling method. In this paper, we report on a straightforward thioarylation of azobenzene derivatives with glycosyl thiols as well as other thiols, thereby increasing the scope of azobenzene conjugation. Even challenging unsymmetrical conjugates can be achieved in good yields via sequential or one-pot procedures. Importantly, red-shifted azoswitches, which are addressed with visible light, were easily functionalized. Additionally, by oxidation of the sulfide bridge to the respective sulfones, both the photochromic and the thermal relaxation properties of the core azobenzene can be tuned. Utilizing this option, we realized orthogonal three-state photoswitching in mixtures containing two distinct azobenzene thioglycosides.     

Unravelling Enzymatic Features in a Supramolecular Iridium Catalyst by Computational Calculations

Non-biological catalysts following the governing principles of enzymes are attractive systems to disclose unprecedented reactivities. Most of those existing catalysts feature an adaptable molecular recognition site for substrate binding that are prone to undergo conformational selection pathways. Herein, we present a non-biological catalyst that is able to bind substrates via the induced fit model according to in-depth computational calculations. The system, which is constituted by an inflexible substrate-recognition site derived from a zinc-porphyrin in the second coordination sphere, features destabilization of ground states as well as stabilization of transition states for the relevant iridium-catalyzed C−H bond borylation of pyridine. In addition, this catalyst appears to be most suited to tightly bind the transition state rather than the substrate. Besides these features, which are reminiscent of the action modes of enzymes, new elementary catalytic steps (i. e. C−B bond formation and catalyst regeneration) have been disclosed owing to the unique distortions encountered in the different intermediates and transition states.     

Chimera Spectrum Diagnostics for Peptides Using Two-Dimensional Partial Covariance Mass Spectrometry

The rate of successful identification of peptide sequences by tandem mass spectrometry (MS/MS) is adversely affected by the common occurrence of co-isolation and co-fragmentation of two or more isobaric or isomeric parent ions. This results in so-called `chimera spectra’, which feature peaks of the fragment ions from more than a single precursor ion. The totality of the fragment ion peaks in chimera spectra cannot be assigned to a single peptide sequence, which contradicts a fundamental assumption of the standard automated MS/MS spectra analysis tools, such as protein database search engines. This calls for a diagnostic method able to identify chimera spectra to single out the cases where this assumption is not valid. Here, we demonstrate that, within the recently developed two-dimensional partial covariance mass spectrometry (2D-PC-MS), it is possible to reliably identify chimera spectra directly from the two-dimensional fragment ion spectrum, irrespective of whether the co-isolated peptide ions are isobaric up to a finite mass accuracy or isomeric. We introduce ‘3-57 chimera tag’ technique for chimera spectrum diagnostics based on 2D-PC-MS and perform numerical simulations to examine its efficiency. We experimentally demonstrate the detection of a mixture of two isomeric parent ions, even under conditions when one isomeric peptide is at one five-hundredth of the molar concentration of the second isomer.