Functional association of the N‐terminal residues with the central region in glucagon‐related peptides

GLP‐1 is an incretin peptide involved in the regulation of glucose metabolism and the glucose‐dependent stimulation of insulin secretion. Ex‐4 is a paralog of GLP‐1 that has comparable GLP‐1R potency but extended physiological action. GLP‐1 and Ex‐4 are helical peptides that share ～50% sequence homology but differ at several residues, notably the second amino acid which controls susceptibility to DPP‐IV cleavage. This single amino acid difference yields divergent receptor potency when studied in the context of the two hormone sequences. Ex‐4 uniquely tolerates Gly2 through select amino acid differences in the middle region of the peptide that are absent in GLP‐1. We report that substitution of Ex‐4 amino acids Glu16, Leu21, and Glu24 to the GLP‐1 sequence enabled Gly2 tolerance. The coordination of the N‐terminus with these central residues shows an interaction of substantial importance not only to DPP‐IV stability but also to receptor activation. Extension of this observation to glucagon‐based co‐agonist peptides showed different structural requirements for effective communication between the N‐terminus and the mid‐section of these peptides in achieving high potency agonism at the GLP‐1 and GCGRs. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Epitope motif of an anti‐nitrotyrosine antibody specific for tyrosine‐nitrated peptides revealed by a combination of affinity approaches and mass spectrometry

Nitration of tyrosine residues has been shown to be an important oxidative modification in proteins and has been suggested to play a role in several diseases such as atherosclerosis, asthma, lung and neurodegenerative diseases. Detection of nitrated proteins has been mainly based on the use of nitrotyrosine‐specific antibodies. In contrast, only a small number of nitration sites in proteins have been unequivocally identified by MS. We have used a monoclonal 3‐NT‐specific antibody, and have synthesized a series of tyrosine‐nitrated peptides of prostacyclin synthase (PCS) in which a single specific nitration site at Tyr‐430 had been previously identified upon reaction with peroxynitrite 17 . The determination of antibody‐binding affinity and specificity of PCS peptides nitrated at different tyrosine residues (Tyr‐430, Tyr‐421, Tyr‐83) and sequence mutations around the nitration sites provided the identification of an epitope motif containing positively charged amino acids (Lys and/or Arg) N‐terminal to the nitration site. The highest affinity to the anti‐3NT‐antibody was found for the PCS peptide comprising the Tyr‐430 nitration site with a K_D of 60 nM determined for the peptide, PCS(424‐436‐Tyr‐430NO₂); in contrast, PCS peptides nitrated at Tyr‐421 and Tyr‐83 had substantially lower affinity. ELISA, SAW bioaffinity, proteolytic digestion of antibody‐bound peptides and affinity‐MS analysis revealed highest affinity to the antibody for tyrosine‐nitrated peptides that contained positively charged amino acids in the N‐terminal sequence to the nitration site. Remarkably, similar N‐terminal sequences of tyrosine‐nitration sites have been recently identified in nitrated physiological proteins, such as eosinophil peroxidase and eosinophil‐cationic protein. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Cellular and viral peptides bind multiple sites on the N‐terminal domain of clathrin

Short peptide motifs in unstructured regions of clathrin‐adaptor proteins recruit clathrin to membranes to facilitate post‐Golgi membrane transport. Three consensus clathrin‐binding peptide sequences have been identified and structural studies show that each binds distinct sites on the clathrin heavy chain N‐terminal domain (NTD). A fourth binding site for adaptors on NTD has been functionally identified but not structurally characterised. We have solved high resolution structures of NTD bound to peptide motifs from the cellular clathrin adaptors β2 adaptin and amphiphysin plus a putative viral clathrin adaptor, hepatitis D virus large antigen (HDAg‐L). Surprisingly, with each peptide we observe simultaneous peptide binding at multiple sites on NTD and viral peptides binding to the same sites as cellular peptides. Peptides containing clathrin‐box motifs (CBMs) with the consensus sequence LΦxΦ[DE] bind at the ‘arrestin box’ on NTD, between β‐propeller blades 4 and 5, which had previously been thought to bind a distinct consensus sequence. Further, we structurally define the fourth peptide binding site on NTD, which we term the Royle box. In vitro binding assays show that clathrin is more readily captured by cellular CBMs than by HDAg‐L, and site‐directed mutagenesis confirms that multiple binding sites on NTD contribute to efficient capture by CBM peptides.      

An HPLC‐ESMS study on the solid‐phase assembly of C‐terminal proline peptides

DKP formation is a serious side reaction during the solid‐phase synthesis of peptide acids containing either Pro or Gly at the C‐terminus. This side reaction not only leads to a lower overall yield, but also to the presence in the reaction crude of several deletion peptides lacking the first amino acids. For the preparation of protected peptides using the Fmoc/tBu strategy, the use of a ClTrt‐Cl‐resin with a limited incorporation of the C‐terminal amino acid is the method of choice. The use of resins with higher loading levels leads to more impure peptide crudes. The use of HPLC‐ESMS is a useful method for analysing complex samples, such as those formed when C‐terminal Pro peptides are prepared by non‐optimized solid‐phase strategies. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and application of Nα‐Fmoc‐Nπ‐4‐methoxybenzyloxymethylhistidine in solid phase peptide synthesis

The 4‐methoxybenzyloxymethyl (MBom) group was introduced at the N^π‐position of the histidine (His) residue by using a regioselective procedure, and its utility was examined under standard conditions used for the conventional and the microwave (MW)‐assisted solid phase peptide synthesis (SPPS) with 9‐fluorenylmethyoxycarbonyl (Fmoc) chemistry. The N^π‐MBom group fulfilling the requirements for the Fmoc strategy was found to prevent side‐chain‐induced racemization during incorporation of the His residue even in the case of MW‐assisted SPPS performed at a high temperature. In particular, the MBom group proved to be a suitable protecting group for the convergent synthesis because it remains attached to the imidazole ring during detachment of the protected His‐containing peptide segments from acid‐sensitive linkers by treatment with a weak acid such as 1% trifluoroacetic acid in dichloromethane. We also demonstrated the facile synthesis of Fmoc‐His(π‐MBom)‐OH with the aid of purification procedure by crystallization to effectively remove the undesired τ‐isomer without resorting to silica gel column chromatography. This means that the present synthetic procedure can be used for large‐scale production without any obstacles. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Evaluation of lipid‐binding properties of the N‐terminal helical segments in human apolipoprotein A‐I using fragment peptides

Although the N‐terminal region in human apolipoprotein (apo) A‐I is thought to stabilize the lipid‐free structure of the protein, its role in lipid binding is unknown. Using synthetic fragment peptides, we examined the lipid‐binding properties of the first 43 residues (1–43) of apoA‐I in comparison with residues 44–65 and 220–241, which have strong lipid affinity in the molecule. Circular dichroism measurements demonstrated that peptides corresponding to each segment have potential propensity to form α‐helical structure in trifluoroethanol. Spectroscopic and thermodynamic measurements revealed that apoA‐I (1–43) peptide has the strong ability to bind to lipid vesicles and to form α‐helical structure comparable to apoA‐I (220–241) peptide. Substitution of Tyr‐18 located at the center of the most hydrophobic region in residues 1–43 with a helix‐breaking proline resulted in the impaired lipid binding, indicating that the α‐helical structure in this region is required to trigger the lipid binding. In contrast, apoA‐I (44–65) peptide exhibited a lower propensity to form α‐helical structure upon binding to lipid, and apoA‐I (44–65/S55P) peptide exhibited diminished, but not completely impaired, lipid binding, suggesting that the central region of residues 44–65 is not pivotally involved in the formation of the α‐helical structure and lipid binding. These results indicate that the most N‐terminal region of apoA‐I molecule, residues 1–43, contributes to the lipid interaction of apoA‐I through the hydrophobic helical residues. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of peptides containing 2‐oxohistidine residues and their characterization by liquid chromatography‐tandem mass spectrometry

Protein oxidation by reactive oxygen species has been associated with aging and neurodegenerative disorders, and histidine is one of the major oxidation targets due to its metal‐chelating property and susceptibility to metal‐catalyzed oxidation. 2‐Oxohistidine, the major product of histidine oxidation, has been recently identified as a stable marker of oxidative damage in biological systems, but its biophysical and biochemical properties are understudied, partly because of difficulties in its chemical synthesis. We developed an efficient method to generate a 2‐oxohistidine side chain using metal‐catalyzed oxidation, applicable to both monomers and peptides. By optimizing reagent ratios and pH buffering in Cu²⁺/ascorbate/O₂ reaction system, we improved the yield more than tenfold compared to reported conditions, which allowed us to obtain homogeneously modified 2‐oxohisidine peptides for further studies. Analysis of 2‐oxohistidine‐containing model peptides by liquid chromatography‐tandem mass spectrometry demonstrated increased retention time in reverse‐phase chromatography and general stability of 2‐oxohistidine under electrospray ionization and collision‐induced dissociation. Thus, large‐scale analysis of 2‐oxohistidine‐modified proteome should be feasible using shotgun protein mass spectrometry, and we were able to observe such peptides in proteomics datasets. The feasibility of acquiring purified peptide probes and peptide antigens containing 2‐oxohistidine will help advance the study of this non‐enzymatic posttranslational modification. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

N‐terminal diproline and charge group effects on the stabilization of helical conformation in alanine‐based short peptides: CD studies with water and methanol as solvent

Protein folding problem remains a formidable challenge as main chain, side chain and solvent interactions remain entangled and have been difficult to resolve. Alanine‐based short peptides are promising models to dissect protein folding initiation and propagation structurally as well as energetically. The effect of N‐terminal diproline and charged side chains is assessed on the stabilization of helical conformation in alanine‐based short peptides using circular dichroism (CD) with water and methanol as solvent. A1 (Ac–Pro–Pro–Ala–Lys–Ala–Lys–Ala–Lys–Ala–NH₂) is designed to assess the effect of N‐terminal homochiral diproline and lysine side chains to induce helical conformation. A2 (Ac–Pro–Pro–Glu–Glu–Ala–Ala–Lys–Lys–Ala–NH₂) and A3 (Ac–d Pro–Pro–Glu–Glu–Ala–Ala–Lys–Lys–Ala–NH₂) with N‐terminal homochiral and heterochiral diproline, respectively, are designed to assess the effect of Glu...Lys (i , i  + 4) salt bridge interactions on the stabilization of helical conformation. The CD spectra of A1 , A2 and A3 in water manifest different amplitudes of the observed polyproline II (PPII) signals, which indicate different conformational distributions of the polypeptide structure. The strong effect of solvent substitution from water to methanol is observed for the peptides, and CD spectra in methanol evidence A2 and A3 as helical folds. Temperature‐dependent CD spectra of A1 and A2 in water depict an isodichroic point reflecting coexistence of two conformations, PPII and β‐strand conformation, which is consistent with the previous studies. The results illuminate the effect of N‐terminal diproline and charged side chains in dictating the preferences for extended‐β, semi‐extended PPII and helical conformation in alanine‐based short peptides. The results of the present study will enhance our understanding on stabilization of helical conformation in short peptides and hence aid in the design of novel peptides with helical structures. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Evaluation of the Edman degradation product of vancomycin bonded to core‐shell particles as a new HPLC chiral stationary phase

A modified macrocyclic glycopeptide‐based chiral stationary phase (CSP), prepared via Edman degradation of vancomycin, was evaluated as a chiral selector for the first time. Its applicability was compared with other macrocyclic glycopeptide‐based CSPs: TeicoShell and VancoShell. In addition, another modified macrocyclic glycopeptide‐based CSP, NicoShell, was further examined. Initial evaluation was focused on the complementary behavior with these glycopeptides. A screening procedure was used based on previous work for the enantiomeric separation of 50 chiral compounds including amino acids, pesticides, stimulants, and a variety of pharmaceuticals. Fast and efficient chiral separations resulted by using superficially porous (core‐shell) particle supports. Overall, the vancomycin Edman degradation product (EDP) resembled TeicoShell with high enantioselectivity for acidic compounds in the polar ionic mode. The simultaneous enantiomeric separation of 5 racemic profens using liquid chromatography‐mass spectrometry with EDP was performed in approximately 3 minutes. Other highlights include simultaneous liquid chromatography separations of rac‐amphetamine and rac‐methamphetamine with VancoShell, rac‐pseudoephedrine and rac‐ephedrine with NicoShell, and rac‐dichlorprop and rac‐haloxyfop with TeicoShell.     

Controlling the helical screw sense of peptides with C‐terminal L‐valine

One chiral L ‐valine (L ‐Val) was inserted into the C‐terminal position of achiral peptide segments constructed from α‐aminoisobutyric acid (Aib) and α,β‐dehydrophenylalanine (Δ^ZPhe) residues. The IR, ¹H NMR and CD spectra indicated that the dominant conformations of the pentapeptide Boc‐Aib‐ΔPhe‐(Aib)₂‐L ‐Val‐NH‐Bn (3) and the hexapeptide Boc‐Aib‐ΔPhe‐(Aib)₃‐L ‐Val‐NH‐Bn (4) in solution were both right‐handed (P) 3₁₀‐helical structures. X‐ray crystallographic analyses of 3 and 4 revealed that only a right‐handed (P) 3₁₀‐helical structure was present in their crystalline states. The conformation of 4 was also studied by molecular‐mechanics calculations. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Identification of peptide‐binding sites within BSA using rapid,laser‐induced covalent cross‐linking combined with high‐performance mass spectrometry

We are developing a rapid, time‐resolved method using laser‐activated cross‐linking to capture protein‐peptide interactions as a means to interrogate the interaction of serum proteins as delivery systems for peptides and other molecules. A model system was established to investigate the interactions between bovine serum albumin (BSA) and 2 peptides, the tridecapeptide budding‐yeast mating pheromone (α‐factor) and the decapeptide human gonadotropin‐releasing hormone (GnRH). Cross‐linking of α‐factor, using a biotinylated, photoactivatable p‐benzoyl‐L‐phenylalanine (Bpa)–modified analog, was energy‐dependent and achieved within seconds of laser irradiation. Protein blotting with an avidin probe was used to detect biotinylated species in the BSA‐peptide complex. The cross‐linked complex was trypsinized and then interrogated with nano‐LC–MS/MS to identify the peptide cross‐links. Cross‐linking was greatly facilitated by Bpa in the peptide, but some cross‐linking occurred at higher laser powers and high concentrations of a non‐Bpa–modified α‐factor. This was supported by experiments using GnRH, a peptide with sequence homology to α‐factor, which was likewise found to be cross‐linked to BSA by laser irradiation. Analysis of peptides in the mass spectra showed that the binding site for both α‐factor and GnRH was in the BSA pocket defined previously as the site for fatty acid binding. This model system validates the use of laser‐activation to facilitate cross‐linking of Bpa‐containing molecules to proteins. The rapid cross‐linking procedure and high performance of MS/MS to identify cross‐links provides a method to interrogate protein‐peptide interactions in a living cell in a time‐resolved manner.     

A new approach for detecting C‐terminal amidation of proteins and peptides by mass spectrometry in conjunction with chemical derivatization

We describe a mass spectrometric method for distinguishing between free and modified forms of the C‐terminal carboxyl group of peptides and proteins, in combination with chemical approaches for the isolation of C‐terminal peptides and site‐specific derivatization of the C‐terminal carboxyl group. This method could most advantageously be exploited to discriminate between peptides having C‐terminal carboxyl groups in the free (COOH) and amide (CONH₂) forms by increasing their mass difference from 1 to 14 Da by selectively converting the free carboxyl group into methylamide (CONHCH₃). This method has been proven to be applicable to peptides containing aspartic and glutamic acids, because all the carboxyl groups except the C‐terminal one are inert to derivatization, according to oxazolone chemistry. The efficiency of the method is illustrated by a comparison of the peaks of processed peptides obtained from a mixture of adrenomedullin, calcitonin, and BSA. Among these components of the mixture, only the C‐terminal peptide of BSA exhibited the mass shift of 13 Da upon treatment, eventually unambiguously validating the C‐terminal amide structures of adrenomedullin and calcitonin. The possibility of extending this method for the analysis of C‐terminal PTMs is also discussed.     

Chaperone‐like activities of different molecular forms of β‐casein. Importance of polarity of N‐terminal hydrophilic domain

As a member of intrinsically unstructured protein family, β‐casein (β‐CN) contains relatively high amount of prolyl residues, adopts noncompact and flexible structure and exhibits chaperone‐like activity in vitro. Like many chaperones, native β‐CN does not contain cysteinyl residues and exhibits strong tendencies for self‐association. The chaperone‐like activities of three recombinant β‐CNs wild type (WT) β‐CN, C4 β‐CN (with cysteinyl residue in position 4) and C208 β‐CN (with cysteinyl residue in position 208), expressed and purified from E. coli, which, consequently, lack the phosphorylated residues, were examined and compared with that of native β‐CN using insulin and alcohol dehydrogenase as target/substrate proteins. The dimers (β‐CND) of C4‐β‐CN and C208 β‐CN were also studied and their chaperone‐like activities were compared with those of their monomeric forms. Lacking phosphorylation, WT β‐CN, C208 β‐CN, C4 β‐CN and C4 β‐CND exhibited significantly lower chaperone‐like activities than native β‐CN. Dimerization of C208 β‐CN with two distal hydrophilic domains considerably improved its chaperone‐like activity in comparison with its monomeric form. The obtained results demonstrate the significant role played by the polar contributions of phosphorylated residues and N‐terminal hydrophilic domain as important functional elements in enhancing the chaperone‐like activity of native β‐CN. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 623–632, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

QCMAP: An Interactive Web‐Tool for Performance Diagnosis and Prediction of LC‐MS Systems

The increasing role played by liquid chromatography‐mass spectrometry (LC‐MS)‐based proteomics in biological discovery has led to a growing need for quality control (QC) on the LC‐MS systems. While numerous quality control tools have been developed to track the performance of LC‐MS systems based on a pre‐defined set of performance factors (e.g., mass error, retention time), the precise influence and contribution of the performance factors and their generalization property to different biological samples are not as well characterized. Here, a web‐based application (QCMAP) is developed for interactive diagnosis and prediction of the performance of LC‐MS systems across different biological sample types. Leveraging on a standardized HeLa cell sample run as QC within a multi‐user facility, predictive models are trained on a panel of commonly used performance factors to pinpoint the precise conditions to a (un)satisfactory performance in three LC‐MS systems. It is demonstrated that the learned model can be applied to predict LC‐MS system performance for brain samples generated from an independent study. By compiling these predictive models into our web‐application, QCMAP allows users to benchmark the performance of their LC‐MS systems using their own samples and identify key factors for instrument optimization. QCMAP is freely available from: http://shiny.maths.usyd.edu.au/QCMAP/ .     

Reductive Alkaline Release of N‐Glycans Generates a Variety of Unexpected,Useful Products

Release of O‐glycans by reductive β‐elimination has become routine in many glyco‐analytical laboratories and concomitant release of N‐glycans has repeatedly been observed. Revisiting this somewhat forgotten mode of N‐glycan release revealed that all kinds of N‐glycans including oligomannosidic and complex‐type N‐glycans from plants with 3‐linked fucose and from mammals with or without 6‐linked fucose and with sialic acid could be recovered. However, the mass spectra of the obtained products revealed very surprising facts. Even after 16 h incubation in 1 M sodium borohydride, a large part of the glycans occurred in reducing form. Moreover, about one third emerged in the form of the stable amino‐functionalized 1‐amino‐1‐deoxy‐glycitol. When avoiding acidic conditions, considerable amounts of glycosylamine were observed. In addition, a compound with a reduced asparagine and de‐N‐acetylation products, in particular of sialylated glycans, was seen. The relative yields of the products reducing glycosylamine, reducing N‐glycan, 1‐amino‐1‐deoxy‐glycitol or glycitol could be controlled by the release conditions, foremost by temperature and borohydride concentration. Thus, chemical release of N‐glycans constitutes a cost‐saving alternative to enzymatic hydrolysis for the preparation of precursors for the production of reference compounds for various formats of N‐glycan analysis. Moreover, it allows to obtain a stable amino‐functionalized glycan derivative, which can be employed to construct glycan arrays or affinity matrices.