Synthesis and characterization of human α‐defensins 4‐6

Abstract: Human α‐defensins are small, Cys‐rich, cationic proteins expressed predominantly in neutrophils and intestinal epithelia. They play important roles in innate and adaptive immunity against infection. Progress in studying these molecules can be accelerated by access to large quantities of high‐quality materials, which have been obtained mainly from natural sources. Here, we report total synthesis of human α‐defensins 4, 5, and 6, also known as HNP4, HD5, and HD6, using the optimized N,N‐diisopropylethylamine (DIEA) in situ neutralization/2‐(1 H‐benzotriazolyl)‐1,1,3,3‐tetramethyluroniumhexafluorophosphate (HBTU) activation protocol for solid‐phase Boc chemistry. Oxidative folding/disulfide formation was achieved directly using crude peptides, resulting in an overall synthetic yield of 10–16% with high purity. Antimicrobial activity assays were performed with Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213, using colony‐counting methods, and the results demonstrated differential activity against these strains. Our report describes a highly efficient synthetic approach that enables thorough structural and functional studies of these three important immunologic molecules.     

A peptide derived from the C‐terminal part of a plant cysteine protease folds into a stack of two β‐hairpins,a scaffold present in the emerging family of granulin‐like growth factors

Abstract: A 35 amino acid residue peptide corresponding to the N‐terminal subdomain of the granulin‐like repeat from rice oryzain β was synthesized and regioselectively oxidized to produce a species with a [1–3, 2–4] disulfide‐pairing pattern. The resulting peptide was studied in solution using NMR and was shown to adopt the tertiary topology of a stack of two β‐hairpins found in the emerging family of granulin‐like growth factors. Because of the longer second β‐hairpin, the overall conformation of the peptide is somewhat more flexible than that of its well‐structured carp granulin‐1 analog. Except for the cysteine alignment, there is very little sequence homology between granulin‐like growth factors from the animal kingdom and the granulin‐like repeats at the C‐termini of plant cysteine proteases. Therefore, the stack of two β‐hairpins may be a conserved three‐dimensional organization of the granulin‐like repeats from evolutionary distant sources with a significant role in specific protein–protein interactions.     

Mapping the receptor binding regions of human chorionic gonadotropin (hCG) using disulfide peptides of its β‐subunit: possible involvement of the disulfide bonds Cys9−Cys57 and Cys23−Cys72 in receptor binding of the hormone

Abstract: Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone essential for the establishment and maintenance of pregnancy. The α‐ and β‐subunits of hCG are highly cross‐linked internally by disulfide bonds which seem to stabilize the tertiary structures required for the noncovalent association of the subunits to generate hormonal activity. The purpose of this study was to delineate the role of the disulfide bonds of hCGβ in receptor binding of the hormone. Six disulfide peptides incorporating each of the six disulfide bonds of hCGβ were synthesized and screened, along with their linear counterparts, for their ability to competitively inhibit the binding of [¹²⁵I] hCG to sheep ovarian corpora luteal LH/CG receptor. Disulfide peptide Cys (9?57) was found to be ≈ 4‐fold more potent than the most active of its linear counterparts in inhibiting radiolabeled hCG from binding to its receptor. Similarly, disulfide peptide Cys (23?72) exhibited receptor binding inhibition activity, whereas the constituent linear peptides were found to be inactive. The results suggest the involvement of the disulfide bonds Cys⁹?Cys⁵⁷ and Cys²³?Cys⁷² of the β‐subunit of hCG in receptor binding of the hormone. This study is the first of its kind to use disulfide peptides rather than linear peptides to map the receptor binding regions of hCG.     

The Proteolytic Stability and Cytotoxicity Studies of l‐Aspartic Acid and l‐Diaminopropionic Acid derived β‐Peptides and a Mixed α/β‐Peptide

The use of peptides as drugs in pharmaceutical applications is hindered by their susceptibility to proteolysis and therefore low bioavailability. β‐Peptides that contain an additional methylene group in the backbone, are gaining recognition from a pharmaceutical stand point as they are considerably more resilient to proteolysis and metabolism. Recently, we reported two new classes of β ‐peptides, β ³‐ and β²‐peptides derived from l ‐aspartic acid and l ‐diaminopropionic acid, respectively. Here, we report the proteolytic stability of these β‐peptidic compounds and a mixed α /β‐peptide against three enzymes (pronase, trypsin and elastase), as well as, human serum. The stability of these peptides was compared to an α‐peptide. Peptides containing β‐linkages were resistant to all conditions. The mixed α /β‐peptide, however, exhibited proteolysis in the presence of trypsin and pronase but not elastase. The rate of degradation of the mixed α /β‐peptide was slower than that would be expected for an α‐peptide. In addition, these β‐peptides were not toxic to HeLa and COS‐1 cell lines as observed by MTT cytotoxicity assay. These results expand the scope of mixed α /β‐peptides containing β‐amino acids or small β‐peptide fragments as therapeutic peptides.     

Solid‐phase synthesis of peptides containing the spin‐labeled 2,2,6,6‐tetramethylpiperidine‐1‐oxyl‐4‐amino‐4‐carboxylic acid (TOAC)

Abstract: 2,2,6,6‐Tetramethylpiperidine‐1‐oxyl‐4‐amino‐4‐carboxylic acid (TOAC) is a nitroxide spin‐labeled, achiral C^α‐tetrasubstituted amino acid recently shown to be not only an effective β‐turn and 3₁₀/α‐helix promoter in peptides, but also an excellent rigid electron paramagnetic resonance probe and fluorescence quencher. Here, we demonstrate that TOAC can be effectively incorporated into internal positions of peptide sequences using Fmoc chemistry and solid‐phase synthesis in an automated apparatus.     

Antibacterial activities and conformations of synthetic α‐defensin HNP‐1 and analogs with one,two and three disulfide bridges

Abstract: Structure and biological activities of synthetic peptides corresponding to human α‐defensin HNP‐1, AC¹YC²RIPAC³IAGERRYGTC⁴IYQGRLWAFC⁵C⁶ with the S–S connectivities: C¹–C⁶, C²–C⁴, C³–C⁵, and its variants with one, two and three disulfide bridges were investigated. Oxidation of synthetic, reduced HNP‐1 yielded a peptide with S–S connectivities C¹–C³, C²–C⁴ and C⁵–C⁶, and not with the S–S linkages as in naturally occurring HNP‐1. Selective protection of cysteine sulfhydryls was necessary for the formation of S–S bridges as in native HNP‐1. Likewise, oxidation of peptide encompassing the segment from C² to C⁵, resulted in the S–S linkages C²–C³ and C⁴–C⁵ instead of the expected linkage C²–C⁴ and C³–C⁵. Antibacterial activities were observed for all peptides, irrespective of how the S–S bridges were linked. Linear peptides without S–S bridges were inactive. Circular dichroism (CD) spectra suggest that peptides constrained by one and two S–S bridges do not form rigid β‐sheet structures in an aqueous environment. The spectrum of HNP‐1 in an aqueous environment suggests the presence of a β‐hairpin conformation. In the presence of lipid vesicles, the S–S constrained peptides tend to adopt a β‐structure. Although the S–S connectivities observed in HNP‐1 may be necessary for other physiological activities, such as chemotaxis, they are clearly not essential for antibacterial activity.     

Insights into the determinants of β‐sheet stability: 1H and 13C NMR conformational investigation of three‐stranded antiparallel β‐sheet‐forming peptides

Abstract: In a previous study we designed a 20‐residue peptide able to adopt a significant population of a three‐stranded antiparallel β‐sheet in aqueous solution (de Alba et al. [1999]Protein Sci. 8, 854–865). In order to better understand the factors contributing to β‐sheet folding and stability we designed and prepared nine variants of the parent peptide by substituting residues at selected positions in its strands. The ability of these peptides to form the target motif was assessed on the basis of NMR parameters, in particular NOE data and ¹³C_α conformational shifts. The populations of the target β‐sheet motif were lower in the variants than in the parent peptide. Comparative analysis of the conformational behavior of the peptides showed that, as expected, strand residues with low intrinsic β‐sheet propensities greatly disfavor β‐sheet folding and that, as already found in other β‐sheet models, specific cross‐strand side chain–side chain interactions contribute to β‐sheet stability. More interestingly, the performed analysis indicated that the destabilization effect of the unfavorable strand residues depends on their location at inner or edge strands, being larger at the latter. Moreover, in all the cases examined, favorable cross‐strand side chain–side chain interactions were not strong enough to counterbalance the disfavoring effect of a poor β‐sheet‐forming residue, such as Gly.     

From pro defensins to defensins: synthesis and characterization of human neutrophil pro α‐defensin‐1 and its mature domain

Abstract: Human neutrophil α‐defensins (HNPs) are small, cationic, Cys‐rich antimicrobial proteins that play important roles in innate immunity against infectious microbes such as bacteria, fungi and enveloped viruses. Synthesized as inactive precursors in vivo (pre‐proHNPs), HNPs are activated through proteolytic removal of the inhibitory pro‐peptide required for subcellular sorting and correct folding. We seek to understand the molecular basis for the recognition between the 45‐residue pro‐peptide and the C‐terminal functional domain. Here we described, total chemical synthesis of the 75‐residue human neutrophil pro α‐defensin‐1 (proHNP1) via native chemical ligation. After oxidative folding, proHNP1 is cleaved by cyanogen bromide at the Met⁴⁵–Ala⁴⁶ peptide bond to release the mature form. The native disulfide connectivity in HNP1, i.e. Cys¹–Cys⁶, Cys²–Cys⁴ and Cys³–Cys⁵, is verified by mass mapping of peptide fragments generated by proteolytic digestion and Edman degradation. Fluorescence spectroscopy studies and antimicrobial activity assays further support that synthetic proHNP1 and HNP1 are correctly folded. While largely unstructured in aqueous solution, the pro‐peptide binds to HNP1 intermolecularly with an apparent K_d value of 6.2 μm at pH 7.4, confirming the mode of intramolecular inactivation of human α‐defensin precursors.     

Effects of increasing hydrophobicity by N‐terminal myristoylation on the antibacterial and hemolytic activities of the C‐terminal cationic segments of human‐β‐defensins 1–3

Analogs of the cationic C‐terminal segments of human‐β‐defensins HBD1‐3, Phd1‐3 with a single disulfide bond, exhibited comparable antimicrobial activity that was salt sensitive. They did not show hemolytic activity. In this study, N‐terminal myristoylation was carried out on Phd1‐3 to examine whether increasing hydrophobicity would result in improved antibacterial activity. The antibacterial activity of the oxidized myristoylated peptides MPhd1‐3 and their reduced forms MPhd1r‐3r was determined. These peptides showed enhanced antibacterial activity as compared to Phd1‐3, on mid‐log phase and stationary phase of Staphylococcus aureus and Escherichia coli, except MPhd1r‐3r that were inactive on stationary‐phase E. coli. In the presence of 150 mm NaCl, MPhd1‐3 showed activity against S. aureus. MPhd1and two exhibited activity against E. coli but MPhd3 was inactive. Zeta potential measurements indicated that MPhd1‐3 were more effective in surface charge neutralization of bacteria as compared to Phd1‐3. MPhd1‐3 exhibited hemolytic activity to varying extents with MPhd1 being most hemolytic. The data indicate that myristoylation enhances antibacterial activity and modulates hemolytic activity to different extents. Apart from hydrophobicity, distribution of cationic residues in MPhd1‐3 plays important roles for these activities.     

Synthesis of carbon‐14–labelled peptides

Carbon‐14 (¹⁴C)–labelled active pharmaceutical ingredients (APIs) and investigational medicinal products (IMPs) are required for phase 0/I to phase III mass balance and micro‐dosing clinical trials. In some cases, this may involve the synthesis of ¹⁴C‐labelled peptides, and the analysis can be performed by accelerated mass spectrometry (AMS). The ¹⁴C‐peptide is typically prepared by the solid‐phase peptide synthesis (SPPS) approach using custom‐made glassware for the key coupling steps. Further modification of the purified ¹⁴C‐peptide can then be performed.     

β‐turn formation by a six‐residue linear peptide in solution

Abstract: A model peptide AAGDYY‐NH₂ (B1), which is found to adopt a β‐turn conformation in the TEM‐1 β‐lactamase inhibitor protein (BLIP) in the TEM‐1/BLIP co‐crystal, was synthesized to elucidate the mechanism of its β‐turn formation and stability. Its structural preferences in solution were comprehensively characterized using CD, FT‐IR and ¹H NMR spectroscopy, respectively. The set of observed diagnostic NOEs, the restrained molecular dynamics simulation, CD and FT‐IR spectroscopy confirmed the formation of a β‐turn in solution by the model peptide. The dihedral angles [(φ₃, ?₃) (φ₄, ?₄)] of [(?52°, ?32°) (?38°, ?44°)] of Gly‐Asp fragment in the model peptide are consistent with those of a type III β‐turn. In a conclusion, the conformational preference of the linear hexapeptide B1 in solution was determined, and it would provide a simple template to study the mechanism of β‐turn formation and stability.     

Structure‐based derivation of peptide inhibitors to target TGF‐β1 receptor for the suppression of hypertrophic scarring fibroblast activation

The intermolecular recognition and interaction between human transforming growth factor β‐1 (TGF‐β1) and its cognate receptor TβRII have been implicated in the pathological condition of hypertrophic scarring (HS). Here, we attempted to rationally derive peptide inhibitors from the complex interface of TGF‐β1 with TβRII to disrupt such interaction for the suppression of fibroblast activation involved in HS. A synthetic strategy that integrated computational design and fluorescence‐based assay was described to examine the structural basis and energetic property of TGF‐β1–TβRII crystal structure, from which a small peptide segment in the complex binding site was stripped artificially. Molecular dynamics simulations revealed that the linear peptide possesses a large intrinsic disorder that would incur considerable entropy penalty upon binding to TβRII; the peptide segment was then extended and cyclized by introducing a disulfide bond across its terminal residues that were premutated to cysteine. Normal mode analysis indicated that, as expected, the peptide flexibility was largely reduced upon the cyclization, and thus, the entropy penalty was minimized substantially, consequently promoting the spontaneous binding of peptide to TβRII. Fluorescence polarization assay confirmed that all linear peptides are typical non‐binders of TβRII (K_d = ND), while the designed cyclic peptides exhibit moderate or high affinity with K_d at micromolar level.     

Conformational specificity of mini‐αA‐crystallin as a molecular chaperone

Abstract: The chaperone activity and biophysical properties of the 19 amino acid peptide DFVIFLDVKHFSPEDLTVK, identified as the functional element in αA‐crystallin and here referred to as mini‐αA‐crystallin, were studied using light scattering and spectroscopic methods after altering its sequence and enantiomerism. The all‐d and all‐l conformers of the peptide do not show marked differences in their chaperone‐like activity against heat‐induced aggregation of alcohol dehydrogenase at 48°C and dithiothreitol‐induced aggregation of insulin. The retro peptide does not show any secondary structure and is also unable to act like a chaperone. Both all‐l and all‐d peptides lose their β‐sheet conformations, hydrophobicity and chaperone‐like activity at temperatures > 50°C. However, upon cooling, a significant portion of those properties was regained, suggesting temperature‐dependent, reversible structural alterations in the peptides under investigation. We propose that both the hydrophobicity and β‐sheet conformation of the functional element of αA‐crystallin are essential for chaperone‐like activity.     

Synthetic peptides derived from the β2−β3 loop of Raphanus sativus antifungal protein 2 that mimic the active site

Abstract: Rs‐AFPs are antifungal proteins, isolated from radish (Raphanus sativus) seed or leaves, which consist of 50 or 51 amino acids and belong to the plant defensin family of proteins. Four highly homologous Rs‐AFPs have been isolated (Rs‐AFP1–4). The structure of Rs‐AFP1 consists of three β‐strands and an α‐helix, and is stabilized by four cystine bridges. Small peptides deduced from the native sequence, still having biological activity, are not only important tools to study structure?function relationships, but may also constitute a commercially interesting target. In an earlier study, we showed that the antifungal activity of Rs‐AFP2 is concentrated mainly in the β2?β3 loop. In this study, we synthesized linear 19‐mer peptides, spanning the entire β2?β3 loop, that were found to be almost as potent as Rs‐AFP2. Cysteines, highly conserved in the native protein, are essential for maintaining the secondary structure of the protein. Surprisingly, in the 19‐mer loop peptides, cysteines can be replaced by α‐aminobutyric acid, which even improves the antifungal potency of the peptides. Analogous cyclic 19‐mer peptides, forced to adopt a hairpin structure by the introduction of one or two non‐native disulfide bridges, were also found to possess high antifungal activity. The synthetic 19‐mer peptides, like Rs‐AFP2 itself, cause increased Ca²⁺ influx in pregerminated fungal hyphae.     

Identification of Fmoc‐β‐Ala‐OH and Fmoc‐β‐Ala‐amino acid‐OH as new impurities in Fmoc‐protected amino acid derivatives*

Abstract: During the manufacture of a proprietary peptide drug substance a new impurity appeared unexpectedly. Investigation of its chemical structure established the impurity as a β‐Ala insertion mutant of the mother peptide. The source of the β‐Ala was identified as contamination of the Fmoc‐Ala‐OH raw material with Fmoc‐β‐Ala‐Ala‐OH. Further studies also demonstrated the presence of β‐Ala in other Fmoc‐amino acids, particularly in Fmoc‐Arg(Pbf)‐OH. In this case, it was due to the presence of both Fmoc‐β‐Ala‐OH and Fmoc‐β‐Ala‐Arg(Pbf)‐OH. It is concluded that β‐Ala contamination of Fmoc‐amino acid derivatives is a general and hitherto unrecognized problem to suppliers of Fmoc‐amino acid derivatives. The β‐Ala is often present as Fmoc‐β‐Ala‐OH and/or as a dipeptide, Fmoc‐β‐Ala‐amino acid‐OH. In collaboration with the suppliers, new specifications were introduced, recognizing the presence of β‐Ala‐related impurities in the raw materials and limiting them to acceptable levels. The implementation of these measures has essentially eliminated β‐Ala contamination as a problem in the manufacture of the drug substance.     

Design and characterization of a membrane permeable N‐methyl amino acid‐containing peptide that inhibits Aβ1–40 fibrillogenesis

Abstract: Alzheimer's disease, Huntington's disease and prion diseases are part of a growing list of diseases associated with formation of β‐sheet containing fibrils. In a previous publication, we demonstrated that the self‐association of the Alzheimer's β‐amyloid (Aβ) peptide is inhibited by peptides homologous to the central core domain of Aβ, but containing N‐methyl amino acids at alternate positions. When these inhibitor peptides are arrayed in an extended, β‐strand conformation, the alternating position of N‐methyl amino acids gives the peptide two distinct faces, one exhibiting a normal pattern of peptide backbone hydrogen bonds, but the other face having limited hydrogen‐bonding capabilities due to the replacement of the amide protons by N‐methyl groups. Here, we demonstrate, through two‐dimensional NMR and circular dichroic spectroscopy, that a pentapeptide with two N‐methyl amino acids, Aβ16–20m or Ac‐K(Me)LV(Me)FF‐NH₂, does indeed have the intended structure of an extended β‐strand. This structure is remarkably stable to changes in solvent conditions and resists denaturation by heating, changes in pH (from 2.5 to 10.5), and addition of denaturants such as urea and guanindine‐HCl. We also show that this peptide, despite its hydrophobic composition, is highly water soluble, to concentrations > 30 mm , in contrast to the nonmethylated congener, Aβ16–20 (Ac‐KLVFF‐NH₂). The striking water solubility, in combination with the hydrophobic composition of the peptide, suggested that the peptide might be able to pass spontaneously through cell membranes and model phospholipid bilayers such as unilamellar vesicles. Thus, we also demonstrate that this peptide is indeed able to pass spontaneously through both synthetic phospholipid bilayer vesicles and cell membranes. Characterization of the biophysical properties of the Aβ16–20m peptide may facilitate the application of this strategy to other systems as diverse as the HIV protease and chemokines, in which there is dimerization through β‐strand domains.     

Role of azaamino acid residue in β‐turn formation and stability in designed peptide

Abstract: The structural perturbation induced by C^αH→N^α exchange in azaamino acid‐containing peptides was predicted by ab initio calculation of the 6‐31G* and 3‐21G* levels. The global energy‐minimum conformations for model compounds, For‐azaXaa‐NH₂ (Xaa = Gly, Ala, Leu) appeared to be the β‐turn motif with a dihedral angle of φ = ± 90°, ψ = 0°. This suggests that incorporation of the azaXaa residue into the i + 2 position of designed peptides could stabilize the β‐turn structure. The model azaLeu‐containing peptide, Boc‐Phe‐azaLeu‐Ala‐OMe, which is predicted to adopt a β‐turn conformation was designed and synthesized in order to experimentally elucidate the role of the azaamino acid residue. Its structural preference in organic solvents was investigated using ¹H NMR, molecular modelling and IR spectroscopy. The temperature coefficients of amide protons, the characteristic NOE patterns, the restrained molecular dynamics simulation and IR spectroscopy defined the dihedral angles [ (φ_i+1, ψ_i+1) (φ_i+2, ψ_i+2)] of the Phe‐azaLeu fragment in the model peptide, Boc‐Phe‐azaLeu‐Ala‐OMe, as [(?59^°, 127^°) (107^°, ?4^°)]. This solution conformation supports a βII‐turn structural preference in azaLeu‐containing peptides as predicted by the quantum chemical calculation. Therefore, intercalation of the azaamino acid residue into the i + 2 position in synthetic peptides is expected to provide a stable β‐turn formation, and this could be utilized in the design of new peptidomimetics adopting a β‐turn scaffold.     

In silico screening and surface plasma resonance‐based verification of programmed death 1‐targeted peptides

Programmed death 1 (PD‐1) is a key immune checkpoint molecule. When it binds to programmed death‐ligand 1 (PD‐L1), it can negatively regulate the immune response. Therefore, blockade of the PD‐1/PD‐L1 interaction could unleash the power of immune system. Though successes achieved by anti‐PD‐1/PD‐L1 antibody drugs in clinical for various cancers, many intrinsic limitations of the high molecular weight drugs require alternatives such as peptide drugs and chemical compounds. In this study, we described a novel in silico approach which was used to screen peptides from PDB database and aimed to identify peptides that have potential to bind the PD‐L1 binding area of PD‐1 molecule. Based on the docking poses, eight peptides were synthesized and measured for their binding abilities by surface plasma resonance technique. The K_D values of the synthesized peptides ranged from 10.0 to 133.0 μM. Furthermore, the binding mechanism between PD‐1 and the peptides was studied. In conclusion, we established a fast and reliable screening method for peptide discovery, which could be applied for identifying peptide inhibitors of various targets. The synthesized peptides could be served as starting points for designing PD‐1 drug for cancer immunotherapy.     

High‐throughput peptide synthesis and peptide purification strategy at the low micromol‐scale using the 96‐well format

Abstract: The increasing demand for short‐ and medium‐sized peptides in many fields of biological, medical and pharmaceutical research requires optimized and universally applicable high‐throughput synthesis and purification techniques at the low‐µmol scale. Here, we describe a continuous peptide synthesis/purification approach using the 96‐well format. First, a µmol scale peptide synthesis on resin beads was optimized on a novel miniaturized 96‐reaction vessel block employing standard Fmoc/tBu‐chemistry. Almost 90% of the synthesized peptides contained the target sequence as the main component, as judged from matrix‐assisted laser desorption/ionization (MALDI) mass spectra. Impurities were mostly related to partially protected peptides. Second, we tested the applicability of ion pair reversed‐phase solid‐phase extraction (IP–RP–SPE) to purify individual peptides. Depending on the length and predicted hydrophobicity of the peptides, elution was performed with 25 or 35% aqueous acetonitrile in the presence of 0.1% trifluoroacetic acid (TFA). Thus, scavengers used during TFA cleavage and partially protected peptides carrying very hydrophobic protecting groups were effectively removed. Using a narrow step gradient, the target peptides were even separated from deleted sequences and protected peptides with similar hydrophobicities. Third, we combined the µmol‐scale synthesis in the 96‐well format with purification by IP–RP–SPE on a 96‐well micro‐extraction plate format. This simple, fast and parallel approach was tested on 12‐mer and 15‐mer peptides to map epitopes of T‐ and B‐cell clones, respectively. Approximately 80% of all peptides were obtained at purities > 90% without purification by RP–HPLC. In summary, this novel approach has several advantages: (i) the µmol‐scale reduced the cost of peptide synthesis, (ii) large numbers of peptides were purified faster, (iii) the volumes of eluents and waste were significantly reduced, and (iv) the RP–HPLC column was not contaminated with hydrophobic impurities.     

Combined use of ESI‐MS and UV diode‐array detection for localization of disulfide bonds in proteins: application to an α‐l‐fucosidase of pea

Abstract: A simplified strategy is described for the assignment of disulfide bonds in proteins of medium to high molecular mass (10–30 kDa). The method combines the use of high‐performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC‐ESI‐MS) and HPLC with UV diode‐array detection (HPLC diode array). The denatured protein is subjected to proteolysis and the peptide mixture is divided into three fractions: (i) underivatized peptides, (ii) ethylpyridylated peptides, and (iii) reduced and ethylpyridylated peptides. The three peptide ensembles are then subjected to chromatographic and spectroscopic analysis. A systematic methodology is described to analyze the large amount of data obtained. The method was applied to the localization of disulfide bonds in α‐l ‐fucosidase from pea. The two disulfide bonds were located between residues Cys⁶⁴ and Cys¹⁰⁹ and between Cys¹⁶² and Cys¹⁶⁹, while Cys¹²⁷ was free.