One‐Pot/Sequential Native Chemical Ligation Using N‐Sulfanylethylanilide Peptide

N‐Sulfanylethylanilide (SEAlide) peptides were developed with the aim of achieving facile synthesis of peptide thioesters by 9‐fluorenylmethyloxycarbonyl (Fmoc)‐based solid‐phase peptide synthesis (Fmoc SPPS). Initially, SEAlide peptides were found to be converted to the corresponding peptide thioesters under acidic conditions. However, the SEAlide moiety was proved to function as a thioester in the presence of phosphate salts and to participate in native chemical ligation (NCL) with N‐terminal cysteinyl peptides, and this has served as a powerful protein synthesis methodology. The reactivity of a SEAlide peptide (anilide vs. thioester) can be easily tuned with or without the use of phosphate salts. This interesting property of SEAlide peptides allows sequential three‐fragment or unprecedented four‐fragment ligation for efficient one‐pot peptide/protein synthesis. Furthermore, dual‐kinetically controlled ligation, which enables three peptide fragments simultaneously present in the reaction to be ligated in the correct order, was first achieved using a SEAlide peptide. Beyond our initial expectations, SEAlide peptides have served in protein chemistry fields as very useful crypto‐peptide thioesters. DOI 10.1002/tcr.201200007     

Fragmentation features of intermolecular cross‐linked peptides using N‐hydroxy‐ succinimide esters by MALDI‐ and ESI‐MS/MS for use in structural proteomics

The use of mass spectrometry coupled with chemical cross‐linking of proteins has become one of the most useful tools for proteins structure and interactions studies. One of the challenges in these studies is the identification of the cross‐linked peptides. The interpretation of the MS/MS data generated in cross‐linking experiments using N‐hydroxy succinimide esters is not trivial once a new amide bond is formed allowing new fragmentation pathways, unlike linear peptides. Intermolecular cross‐linked peptides occur when two different peptides are connected by the cross‐linker and they yield information on the spatial proximity of different domains (within a protein) or proteins (within a complex). In this article, we report a detailed fragmentation study of intermolecular cross‐linked peptides, generated from a set of synthetic peptides, using both ESI and MALDI to generate the precursor ions. The fragmentation features observed here can be helpful in the interpretation and identification of cross‐linked peptides present in cross‐linking experiments and be further implemented in search engine's algorithms. Copyright © 2011 John Wiley & Sons, Ltd.     

Synthesis of Disulfide Surrogate Peptides Incorporating Large‐Span Surrogate Bridges Through a Native‐Chemical‐Ligation‐Assisted Diaminodiacid Strategy

The use of synthetic bridges as surrogates for disulfide bonds has emerged as a practical strategy to obviate the poor stability of some disulfide‐containing peptides. However, peptides incorporating large‐span synthetic bridges are still beyond the reach of existing methods. Herein, we report a native chemical ligation (NCL)‐assisted diaminodiacid (DADA) strategy that enables the robust generation of disulfide surrogate peptides incorporating surrogate bridges up to 50 amino acids in length. This strategy provides access to some highly desirable but otherwise impossible‐to‐obtain disulfide surrogates of bioactive peptide. The bioactivities and structures of the synthetic disulfide surrogates were verified by voltage clamp assays, NMR, and X‐ray crystallography; and stability studies established that the disulfide replacements effectively overcame the problems of disulfide reduction and scrambling that often plague these pharmacologically important peptides.     

Rapid sampling of all‐atom peptides using a library‐based polymer‐growth approach

We adapted existing polymer growth strategies for equilibrium sampling of peptides described by modern atomistic forcefields with a simple uniform dielectric solvent. The main novel feature of our approach is the use of precalculated statistical libraries of molecular fragments. A molecule is sampled by combining fragment configurations—of single residues in this study—which are stored in the libraries. Ensembles generated from the independent libraries are reweighted to conform with the Boltzmann‐factor distribution of the forcefield describing the full molecule. In this way, high‐quality equilibrium sampling of small peptides (4–8 residues) typically requires less than one hour of single‐processor wallclock time and can be significantly faster than Langevin simulations. Furthermore, approximate, clash‐free ensembles can be generated for larger peptides (up to 32 residues in this study) in less than a minute of single‐processor computing. We discuss possible applications of our growth procedure to free energy calculation, fragment assembly protein‐structure prediction protocols, and to “multi‐resolution” sampling. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2011     

Peptide‐Stabilized,Fluorescent Silver Nanoclusters: Solid‐Phase Synthesis and Screening

Few‐atom silver nanoclusters (AgNCs) can exhibit strong fluorescence; however, they require ligands to prevent aggregation into larger nanoparticles. Fluorescent AgNCs in biopolymer scaffolds have so far mainly been synthesized in solution, and peptides have only found limited use compared to DNA. Herein, we demonstrate how solid‐phase methods can increase throughput dramatically in peptide ligand screening and in initial evaluation of fluorescence intensity and chemical stability of peptide‐stabilized AgNCs (P‐AgNCs). 9‐Fluorenylmethyloxycarbonyl (Fmoc) solid‐phase peptide synthesis on a hydroxymethyl‐benzoic acid (HMBA) polyethylene glycol polyacrylamide copolymer (PEGA) resin enabled on‐resin screening and evaluation of a peptide library, leading to identification of novel peptide‐stabilized, fluorescent AgNCs. Using systematic amino acid substitutions, we synthesized and screened a 144‐member library. This allowed us to evaluate the effect of length, charge, and Cys content in peptides used as ligands for AgNC stabilization. The results of this study will enable future spectroscopic studies of these peptide‐stabilized AgNCs for bioimaging and other applications.     

Gas‐Phase Peptide Structures Unraveled by Far‐IR Spectroscopy: Combining IR‐UV Ion‐Dip Experiments with Born–Oppenheimer Molecular Dynamics Simulations

Vibrational spectroscopy provides an important probe of the three‐dimensional structures of peptides. With increasing size, these IR spectra become very complex and to extract structural information, comparison with theoretical spectra is essential. Harmonic DFT calculations have become a common workhorse for predicting vibrational frequencies of small neutral and ionized gaseous peptides. ¹ Although the far‐IR region (<500 cm^?1) may contain a wealth of structural information, as recognized in condensed phase studies, ² DFT often performs poorly in predicting the far‐IR spectra of peptides. Here, Born–Oppenheimer molecular dynamics (BOMD) is applied to predict the far‐IR signatures of two γ‐turn peptides. Combining experiments and simulations, far‐IR spectra can provide structural information on gas‐phase peptides superior to that extracted from mid‐IR and amide A features.     

Electrochemical Study of Tryptophan‐containing Peptides‐Cu(II) Complexes

This article presents rotating ring‐disc electrode investigations of (A = alanine, F = phenylalanine, G = glycine, L = leucine, W = tryptophan) W, GW, WGG, GWG, GGW, GWGG, and GGWA. In addition, the analyses of the copper complexes of the same peptides plus GF, FGG, GFG, FGG, GGFL, GGGG, AAAA, and GGGGGG have been carried out. The results suggest that an influential step in the reaction mechanism of the copper complexes of the tryptophan‐containing peptides (W‐peptides) is the alteration of the peptide structure after the one‐electron oxidation of tryptophan. This change in structure leads to a positive shift in redox potential for the Cu(III)/Cu(II) couple. The analytical implications for the electrochemical detection of W‐peptides as their copper complexes are applied for detection of W‐containing bioactive peptides. Application of the optimized detection conditions of peptides as their Cu(II) complexes are as follows: (1) If sensitivity is paramount, detect the copper complexes at a relatively high potential, around 0.7 V vs. Ag/AgCl. (2) If selectivity is paramount, use a dual electrode detector, oxidize at an upstream anode at 0.4 V, and detect at the downstream cathode at 0–0.1 V.     

Engineering the Ionic Self‐Assembly of Polyoxometalates and Facial‐Like Peptides

The self‐assembly behavior of polyoxometalates (PMs) and facial‐like cationic peptides carrying lysine residues were systematically investigated. Circular dichroism and UV/Vis spectra demonstrated that the multivalent electrostatic attractions between polyanionic PMs and short peptides with protonated lysine residues initiated the conformational transition of peptide molecules from random‐coil to β‐sheet state, and subsequently the co‐assembly. TEM and atomic force microscopy (AFM) measurements showed that uniform nanofibers formed with decreasing size of the PMs or increasing the intermolecular forces of the peptides, such as through hydrogen‐bonding, hydrophobic, and/or π–π interactions. Additionally, the stability of the nanostructures can be improved by rational suppression of the electrostatic repulsion of the shell peptides covering the surface of the nanostructures. These results provide new insight into understanding the ionic self‐assembly of peptides and PMs and controlling their final morphology.     

MiniAp‐4: A Venom‐Inspired Peptidomimetic for Brain Delivery

Drug delivery across the blood–brain barrier (BBB) is a formidable challenge for therapies targeting the central nervous system. Although BBB shuttle peptides enhance transport into the brain non‐invasively, their application is partly limited by lability to proteases. The present study proposes the use of cyclic peptides derived from venoms as an affordable way to circumvent this drawback. Apamin, a neurotoxin from bee venom, was minimized by reducing its complexity, toxicity, and immunogenicity, while preserving brain targeting, active transport, and protease resistance. Among the analogues designed, the monocyclic lactam‐bridged peptidomimetic MiniAp‐4 was the most permeable. This molecule is capable of translocating proteins and nanoparticles in a human‐cell‐based BBB model. Furthermore, MiniAp‐4 can efficiently deliver a cargo across the BBB into the brain parenchyma of mice.     

MeCAT peptide labeling for the absolute quantification of proteins by 2D‐LC‐ICP‐MS

Metal‐Coded Affinity Tags (MeCAT) reagents were devised for the absolute quantification of labeled proteins and peptides using inductively coupled plasma mass spectrometry (ICP‐MS). After the recent publication of quantification approaches for digested proteins, this work presents a multidimensional strategy for the application of MeCAT to samples which require higher chromatographic resolution. Two‐dimensional separations based on strong cation exchange (SCX) and reversed‐phase (RP) chromatography, were used for the quantification of lysozyme, bovine serum albumin and transferrin after tryptic digestion. The elution protocols were optimized to improve the resolution of the MeCAT‐labeled peptides which led to faster elutions in SCX and longer retention times in RP compared with unlabeled peptides. The optimized method provided enough resolution for the samples analyzed. Peptides losses during the whole procedure were studied. Although recoveries of greater than 90% were found in the RP dimension, important global losses in the two‐dimensional offline approach forced us to use specific internal standards, in this case MeCAT‐labeled standard peptides. External calibration and label‐specific isotope dilution analysis (IDA) were tested and compared as possible quantification techniques. While both techniques showed accurate and precise determinations, the label‐specific IDA technique resulted in more straightforward measurements and more affordable external calibrations. Finally, simultaneous quantification of three different samples labeled with different lanthanides was successfully performed demonstrating the potential of MeCAT combined with ICP‐MS for multiplexing. Electrospray ionization mass spectrometry techniques provided the structural information needed for the identification of the labeled species. Copyright © 2012 John Wiley & Sons, Ltd.     

Synthesis and Macrodomain Binding of Mono‐ADP‐Ribosylated Peptides

Mono‐ADP‐ribosylation is a dynamic posttranslational modification (PTM) with important roles in signaling. Mammalian proteins that recognize or hydrolyze mono‐ADP‐ribosylated proteins have been described. We report the synthesis of ADP‐ribosylated peptides from the proteins histone H2B, RhoA and, HNP‐1. An innovative procedure was applied that makes use of pre‐phosphorylated amino acid building blocks. Binding assays revealed that the macrodomains of human MacroD2 and TARG1 exhibit distinct specificities for the different ADP‐ribosylated peptides, thus showing that the sequence surrounding ADP‐ribosylated residues affects the substrate selectivity of macrodomains.     

Cysteine‐reactive covalent capture tags for enrichment of cysteine‐containing peptides

Considering the tremendous complexity and the wide dynamic range of protein samples from biological origin and their proteolytic peptide mixtures, proteomics largely requires simplification strategies. One common approach to reduce sample complexity is to target a particular amino acid in proteins or peptides, such as cysteine (Cys), with chemical tags in order to reduce the analysis to a subset of the whole proteome. The present work describes the synthesis and the use of two new cysteinyl tags, so‐called cysteine‐reactive covalent capture tags (C³T), for the isolation of Cys‐containing peptides. These bifunctional molecules were specifically designed to react with cysteines through iodoacetyl and acryloyl moieties and permit efficient selection of the tagged peptides. To do so, a thioproline was chosen as the isolating group to form, after a deprotection/activation step, a thiazolidine with an aldehyde resin by the covalent capture (CC) method. The applicability of the enrichment strategy was demonstrated on small synthetic peptides as well as on peptides derived from digested proteins. Mass spectrometric (MS) analysis and tandem mass spectrometric (MS/MS) sequencing confirmed the efficient and straightforward selection of the cysteine‐containing peptides. The combination of C³T and CC methods provides an effective alternative to reduce sample complexity and access low abundance proteins. Copyright © 2009 John Wiley & Sons, Ltd.     

Racemic and Quasi‐Racemic X‐ray Structures of Cyclic Disulfide‐Rich Peptide Drug Scaffolds

Cyclic disulfide‐rich peptides have exceptional stability and are promising frameworks for drug design. We were interested in obtaining X‐ray structures of these peptides to assist in drug design applications, but disulfide‐rich peptides can be notoriously difficult to crystallize. To overcome this limitation, we chemically synthesized the L ‐ and D ‐forms of three prototypic cyclic disulfide‐rich peptides: SFTI‐1 (14‐mer with one disulfide bond), cVc1.1 (22‐mer with two disulfide bonds), and kB1 (29‐mer with three disulfide bonds) for racemic crystallization studies. Facile crystal formation occurred from a racemic mixture of each peptide, giving structures solved at resolutions from 1.25 Å to 1.9 Å. Additionally, we obtained the quasi‐racemic structures of two mutants of kB1, [G6A]kB1, and [V25A]kB1, which were solved at a resolution of 1.25 Å and 2.3 Å, respectively. The racemic crystallography approach appears to have broad utility in the structural biology of cyclic peptides.     

Voltammetric Determination of Surface‐Confined Biomolecules with N‐(2‐Ethyl‐ferrocene)maleimide

A thiol‐specific electroactive cross‐linker, N‐(2‐ethyl‐ferrocene)maleimide (Fc‐Mi), has been used to tag surface‐confined peptides containing cysteine residues or oligodeoxynucleotides (ODNs) whose 3′ ends have been modified with thiol groups. The peptides studied herein include both the oxidized and reduced forms of glutathione and a hexapeptide. Cyclic voltammograms (CVs) of the Fc‐Mi groups attached to the surfaces were used to quantify the total number of cysteine residues that are tagged and/or can undergo facile electron transfer reactions with the underlying electrodes. A quartz crystal microbalance was used in conjunction with CV to estimate the total number of cysteine groups labeled by Fc‐Mi per peptide molecule. By comparing to mass spectrometric studies, it is confirmed that not all of the Fc‐Mi linked to the cysteine groups can participate in the electron transfer reactions. The methodology is further extended to the determination of ODN samples in a sandwich assay wherein the thiol linker on the 3′ end can be tagged with Fc‐Mi. The analytical performance was evaluated through determinations of a complementary ODN target and targets with varying numbers of mismatching bases. ODN samples as low as 10 fmol can be detected. Such a low detection level is remarkable considering that no signal amplification scheme is involved in the current method. The approach is shown to be sequence‐ and/or structure‐specific and does not require sophisticated instrumentation and complex experimental procedure.     

MSQ: a tool for quantification of proteomics data generated by a liquid chromatography/matrix‐assisted laser desorption/ionization time‐of‐flight tandem mass spectrometry based targeted quantitative proteomics platform

Mass spectrometry (MS)‐based quantitative proteomics has become a critical component of biological and clinical research for identification of biomarkers that can be used for early detection of diseases. In particular, MS‐based targeted quantitative proteomics has been recently developed for the detection and validation of biomarker candidates in complex biological samples. In such approaches, synthetic reference peptides that are the stable isotope labeled version of proteotypic peptides of proteins to be quantitated are used as internal standards enabling specific identification and absolute quantification of targeted peptides. The quantification of targeted peptides is achieved using the intensity ratio of a native peptide to the corresponding reference peptide whose spike‐in amount is known. However, a manual calculation of the ratios can be time‐consuming and labor‐intensive, especially when the number of peptides to be tested is large. To establish a liquid chromatography/matrix‐assisted laser desorption/ionization time‐of‐flight tandem mass spectrometry (LC/MALDI TOF/TOF)‐based targeted quantitative proteomics pipeline, we have developed a software named Mass Spectrometry based Quantification (MSQ). This software can be used to automate the quantification and identification of targeted peptides/proteins by the MALDI TOF/TOF platform. MSQ was applied to the detection of a selected group of targeted peptides in pooled human cerebrospinal spinal fluid (CSF) from patients with Alzheimer's disease (AD) in comparison with age‐matched control (OC). The results for the automated quantification and identification of targeted peptides/proteins in CSF were in good agreement with results calculated manually. Copyright © 2010 John Wiley & Sons, Ltd.     

Easy‐to‐Attach/Detach Solubilizing‐Tag‐Aided Chemical Synthesis of an Aggregative Capsid Protein

A solubilizing Trt‐K₁₀ tag was developed for the effective chemical preparation of peptides/proteins with low solubility. The Trt‐K₁₀ tag comprises a hydrophilic oligo‐Lys sequence and a trityl anchor, and can be selectively introduced to a side chain thiol of Cys of deprotected peptides/proteins with a trityl alcohol‐type introducing reagent Trt(OH)‐K₁₀ under acidic conditions. Significantly, the ligation product in the reaction mixture of a thiol‐additive‐free native chemical ligation can be modified directly in a one‐pot manner to facilitate the isolation of the product by high‐performance liquid chromatography. Finally, the Trt‐K₁₀ tag can be readily removed with a standard trifluoroacetic acid cocktail. Using this easy‐to‐attach/detach tag‐aided method, a hepatitis B virus capsid protein that is usually difficult to handle was synthesized successfully.     

Virus‐Based Chemical and Biological Sensing

Viruses have recently proven useful for the detection of target analytes such as explosives, proteins, bacteria, viruses, spores, and toxins with high selectivity and sensitivity. Bacteriophages (often shortened to phages), viruses that specifically infect bacteria, are currently the most studied viruses, mainly because target‐specific nonlytic phages (and the peptides and proteins carried by them) can be identified by using the well‐established phage display technique, and lytic phages can specifically break bacteria to release cell‐specific marker molecules such as enzymes that can be assayed. In addition, phages have good chemical and thermal stability, and can be conjugated with nanomaterials and immobilized on a transducer surface in an analytical device. This Review focuses on progress made in the use of phages in chemical and biological sensors in combination with traditional analytical techniques. Recent progress in the use of virus–nanomaterial composites and other viruses in sensing applications is also highlighted.     

Polyoxometalate Clusters Integrated into Peptide Chains and as Inorganic Amino Acids: Solution‐ and Solid‐Phase Approaches

General synthetic methods for the grafting of peptide chains onto polyoxometalate clusters by the use of general activated precursors have been developed. Using a solution‐phase approach, pre‐synthesized peptides can be grafted to a metal oxide cluster to produce hybrids of unprecedented scale (up to 30 residues). An adapted solid‐phase method allows the incorporation of these clusters, which may be regarded as novel hybrid unnatural amino acids, during the peptide synthesis itself. These methods may open the way for the automated synthesis of peptides and perhaps even proteins that contain “inorganic” amino acids.     

Surface‐Modifiable Free‐Floating Films Formed by Multiway Connection of Collagen‐Like Triple‐Helical Peptides

Square‐millimeter‐sized free‐floating translucent films are formed in physiological buffer by multiway connections between biotinylated collagen‐like triple‐helical peptides and avidin. Although the compositions of the films are almost constant, regardless of the ratios of the components loaded, their thicknesses can be controlled by the concentrations of the components. The film surfaces can be further modified by taking advantage of exposed biotin (or avidin) functionalities. The self‐assembled films could serve as novel materials in biomedical and biosensing applications.     

A convenient purification and preconcentration of peptides with α‐cyano‐4‐hydroxycinnamic acid matrix crystals in a pipette tip for matrix‐assisted laser desorption/ionization mass spectrometry

Peptide samples derived from enzymatic in‐gel digestion of proteins resolved by gel electrophoresis often contain high amount of salts originating from reaction and separation buffers. Different methods are used for desalting prior to matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry (MS), e.g. reversed‐phase pipette tip purification, on‐target washing, adding co‐matrices, etc. As a suitable matrix for MALDI MS of peptides, α‐cyano‐4‐hydroxycinnamic acid (CHCA) is frequently used. Crystalline CHCA shows the ability to bind peptides on its surface and because it is almost insoluble in acidic water solutions, the on‐target washing of peptide samples can significantly improve MALDI MS signals. Although the common on‐target washing represents a simple, cheap and fast procedure, only a small portion of the available peptide solution is efficiently used for the subsequent MS analysis. The present approach is a combination of the on‐target washing principle carried out in a narrow‐end pipette tip (e.g. GELoader tip) and preconcentration of peptides from acidified solution by passing it through small CHCA crystals captured inside the tip on a glass microfiber frit. The results of MALDI MS analysis using CHCA‐tip peptide preconcentration are comparable with the use of homemade POROS R2 pipette tip microcolumns. Advantages and limitations of this approach are discussed. Copyright © 2009 John Wiley & Sons, Ltd.