Medium-sized Peptides Research Articles

Microbial degradation of linear peptides by strain B-9 of Sphingosinicella and its application in peptide quantification using liquid chromatography-mass spectrometry

The bacterial strain Sphingosinicella sp. B-9 was originally discovered to have the ability to degrade cyanobacterial cyclic peptides (microcystins), and has three hydrolytic enzymes (MlrA, MlrB, and MlrC). The purpose of this study was to examine in detail the degradation of glucagon/vasoactive intestinal polypeptide (VIP) family peptides by B-9, and to investigate the substrate specificity of B-9 proteases and the possibility of using a B-9 protease as a novel protease for peptide quantification by using a surrogate peptide and mass spectrometry (MS). The effective use of inhibitors revealed the following hydrolytic capability of B-9: One of the B-9 proteases (presumably MlrB) that was not inhibited by ethylenediaminetetraacetic acid (EDTA) cleaved bioactive peptides into medium-sized peptides with broad selectivity, similar to neutral endopeptidase, and another protease that was not inhibited by phenylmethylsulfonyl fluoride (PMSF) corresponded to MlrC and cleaved the resulting medium-sized peptides to smaller peptides or amino acids. The former property was desirable to obtain a suitable surrogate peptide, which was used successfully to quantify peptide using liquid chromatography (LC)-MS. Thus, the present study verified that one of the B-9 proteases has broad cleavage selectivity and cleavage sites, not seen in commercially available proteases, and is applicable to protein and peptide quantification using LC-MS.

Proteolytic and antimicrobial activity of lactic acid bacteria grown in goat milk

We examined 62 strains and 21 trade starter cultures from the collection of LB Bulgaricum PLC for proteolytic and antimicrobial activity of lactic acid bacteria (LAB) grown in goat milk. The aim of this study was to investigate the fermentation of caseins, α-lactalbumin and β-lactoglobulin by LAB, using the o-phthaldialdehyde (OPA) spectrophotometric assay and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The proteolysis targeted mainly caseins, especially β-casein. Whey proteins were proteolyzed, essentially β-lactoglobulin. The proteolytic activity of Lactococcus lactis l598, Streptococcus thermophilus t3D1, Dt1, Lactobacillus lactis 1043 and L. delbrueckii subsp. bulgaricus b38, b122 and b24 was notably high. The proteolysis process gave rise to medium-sized peptide populations. Most of the examined strains showed antimicrobial activity against some food pathogens, such as Escherichia coli, Staphylococcus aureus, Salmonella cholere enteridis, Listeria monocytogenes, Listeria innocua and Enterobacter aerogenes. The most active producers of antimicrobial-active peptides were strains of L. delbrueckii subsp. bulgaricus and S. thermophilus, which are of practical importance. The starter cultures containing the examined species showed high proteolytic and antimicrobial activity in skimmed goat milk. The greatest antimicrobial activity of the cultures was detected against E. aerogenes. The obtained results demonstrated the significant proteolytic potential of the examined strains in goat milk and their potential for application in the production of dairy products from goat's milk. The present results could be considered as the first data on the proteolytic capacity of strains and starter cultures in goat milk for the purposes of trade interest of LB Bulgaricum PLC.

Sequential release of milk protein–derived bioactive peptides in the jejunum in healthy humans

The digestive hydrolysis of dietary proteins leads to the release of peptides in the intestinal tract, where they may exert a variety of functions, but their characterization and quantification are difficult. We aimed to characterize and determine kinetics of the formation of peptides present in the jejunum of humans who ingested casein or whey proteins by using mass spectrometry and to look for and quantify bioactive peptides. Subjects were equipped with a double-lumen nasogastric tube that migrated to the proximal jejunum. A sample collection was performed for 6 h after the ingestion of 30 g (15)N-labeled casein (n = 7) or whey proteins (WPs; n = 6). Nitrogen flow rates were measured, and peptides were identified by using mass spectrometry. After casein ingestion, medium-size peptides (750-1050 kDa) were released during 6 h, whereas larger peptides (1050-1800 kDa) were released from WPs in the first 3 h. A total of 356 and 146 peptides were detected and sequenced in the jejunum after casein and WP ingestion, respectively. β-casein was the most important precursor of peptides, including bioactive peptides with various activities. The amounts of β-casomorphins (β-casein 57-, 58-, 59-, and 60-66) and β-casein 108-113 released on the postprandial window were sufficient to elicit the biological action of these peptides (ie, opioid and antihypertensive, respectively). Clear evidence is shown of the presence of bioactive peptides in the jejunum of healthy humans who ingested casein. Our findings raise the question about the physiologic conditions under which these peptides can express their bioactivity in humans. This trial was registered at clinicaltrials.gov as NCT00862329.

High growth rates in Atlantic salmon (Salmo salar L.) fed 7.5% fish meal in the diet. Micro-, ultra- and nano-filtration of stickwater and effects of different fractions and compounds on pellet quality and fish performance.

The intention of this study is to investigate the effect of fish meal soluble protein quality on growth and protein utilisation of Atlantic salmon fed very low fish meal diets. For this purpose stickwater (SW) was separated during herring meal production, fractionated following successive micro- ultra- and nano-filtration steps and reintroduced in the press cake (PC) in order to produce 4 reconstituted fish meals containing different molecular size soluble protein mixes. The SW fractions used for the production of the experimental fish meals were: 1) the micro-filtration (MF) retentate, 2) the ultra-filtration (UF) retentate, 3) the nano-filtration (NF) retentate and 4) the nano-filtration permeate. Four very low fish meal experimental diets were prepared containing 25gkg−1 super prime fish meal and 50gkg−1 diet of one of the 4 experimental fish meals produced. A positive control diet was prepared containing 300gkg−1 diet super prime fish meal. Three more diets were prepared containing 25gkg−1 diet super prime fish meal and 50gkg−1 diet PC. Two of them were supplemented with either crystalline taurine, or crystalline hydroxyproline, up to the levels present in the high fish meal control diet. The experimental diets with the SW fractions contained similar levels of water soluble protein (WSP), which were higher than in the PC diets and lower than in the control. All diets were iso-nitrogenous, iso-lipidic and iso-energetic and were fed to 8 triplicate groups of salmon (80 fish per group of initial body weight 133g), for 69days. High growth rates and low fish in-fish out ratios (FIFO) were achieved in salmon fed all the very low fish meal diets. Feed intake, fish body growth, whole body protein content, protein gain and morphometry were significantly affected by the inclusion of the different SW fractions in the low fish meal diets. The medium size peptides (UF retentate) induced significant increase in feed consumption in salmon and supported higher fish growth compared to the highest (MF retentate) or the lowest molecular weight peptides (NF permeate) where both feed intake and fish growth were reduced. The fish fed the diet containing the NF permeate had significantly lower growth compared to the diets containing the other SW fractions. We did not find any benefits for salmon performance by the supplementation of taurine or hydroxyproline alone to the low WSP PC diets. The present work gives evidence on the multi-dimensional effects of the different marine water soluble components on fish performance. In order to identify novel, high added-value, water soluble marine compounds, advanced large scale separation technologies must be further developed and applied in studies investigating specific functional effects that these compounds may have on fish and other organisms.

N-to-C Sequential Ligation Using Peptidyl N,N-Bis(2-mercaptoethyl)amide Building Blocks

A novel N- to C-terminus sequential chemical ligation approach has been developed for protein synthesis. Key to this strategy is the relative stability of the N,N-bis(2-mercaptoethyl)amide (BMEA) to the conventional conditions of native chemical ligation. We have also found a new thiol additive for the BMEA-mediated ligation reaction. The usefulness of this approach was demonstrated in the syntheses of a medium-sized peptide and ubiquitin.

Urinary Peptides and Calcium Oxalate Nephrolithiasis, A Combined Approach: MALDI-TOF Mass Spectrometry and MicroBCA Protein Assay

Urinary lithiasis is one of the most common benign urological diseases. There is growing evidence that a delicate equilibrium regulated by the function of proteins, soluble peptides, membrane proteins and intracellular mechanisms actually exists. We have studied the urinary protein composition of patients affected by calcium oxalate nephrolithiasis in order to discover a biomarker or any predisposing factors. The urinary protein composition of 17 patients (11 males, 6 females; mean age 45yrs ± 14SD), affected by calcium oxalate nephrolithiasis, was assessed in comparison with 17 healthy subjects. A qualitative assay was performed using MALDI-TOF mass spectrometry in a spectrum between 1 and 5kDa (medium size peptides), and a numerical (quantitative) assay using specific filters and MicroBCA Protein Assay. No differences were detected in the mass spectrums between patients and control subjects: all peaks overlapped perfectly. The results of the numerical assay suggest that concentrations of protein species <5kDa in control samples were actually higher than those which were found in patients. The differences are statistically significant. The study detected neither a biomarker nor any predisposing factors in "stone former" patients. The assessment of the results obtained, in terms of quantitative differences, indicate the need for further research.

Fully Enzymatic N→C-Directed Peptide Synthesis Using C-Terminal Peptide α-Carboxamide to Ester Interconversion

Chemoenzymatic peptide synthesis is potentially the most cost-efficient technology for the synthesis of short and medium-sized peptides with some important advantages. For instance, stoichiometric amounts of expensive coupling reagents are not required and racemisation does not occur rendering purification easier compared to chemical peptide synthesis. In this paper, a novel interconversion reaction of peptide C-terminal α-carboxamides into primary alkyl esters with alcalase was used to develop a fully enzymatic peptide synthesis strategy. For each elongation step a cost-efficient amino acid carboxamide building block was used followed by the interconversion of the elongated peptide carboxamide to the corresponding primary alkyl ester. These peptide esters are the starting materials for the next enzymatic peptide elongation step.

Enzymatic synthesis of activated esters and their subsequent use in enzyme-based peptide synthesis

Chemoenzymatic peptide synthesis is potentially the most cost-efficient technology for the synthesis of short and medium-sized peptides. However, there are still some limitations when challenging peptides, e.g. containing sterically demanding acyl donors, non-proteinogenic amino acids or proline residues, are to be synthesized. To remedy these limitations, special ester moieties have been used that are specifically recognized by the enzyme, e.g. guanidinophenyl, carboxamidomethyl (Cam) or trifluoroethyl (Tfe) esters, which, unfortunately, are notoriously difficult to synthesize chemically. Herein, we demonstrate that Cam and Tfe esters are very useful for Alcalase-CLEA mediated peptide synthesis using sterically demanding and non-proteinogenic acyl donors as well as poor nucleophiles, and combinations thereof. Furthermore, these esters can be efficiently synthesized by using the lipase Cal-B or Alcalase-CLEA. Finally, it is shown that the ester synthesis by Cal-B and subsequent peptide synthesis by Alcalase-CLEA can be performed simultaneously using a two-enzyme-one-pot approach with glycolamide or 2,2,2-trifluoroethanol as additive.

Advantages of Isotopic Depletion of Proteins for Hydrogen/Deuterium Exchange Experiments Monitored by Mass Spectrometry

Solution-phase hydrogen/deuterium exchange (HDX) monitored by mass spectrometry is an excellent tool to study protein-protein interactions and conformational changes in biological systems, especially when traditional methods such as X-ray crystallography or nuclear magnetic resonance are not feasible. Peak overlap among the dozens of proteolytic fragments (including those from autolysis of the protease) can be severe, due to high protein molecular weight(s) and the broad isotopic distributions due to multiple deuterations of many peptides. In addition, different subunits of a protein complex can yield isomeric proteolytic fragments. Here, we show that depletion of (13)C and/or (15)N for one or more protein subunits of a complex can greatly simplify the mass spectra, increase the signal-to-noise ratio of the depleted fragment ions, and remove ambiguity in assignment of the m/z values to the correct isomeric peptides. Specifically, it becomes possible to monitor the exchange progress for two isobaric fragments originating from two or more different subunits within the complex, without having to resort to tandem mass spectrometry techniques that can lead to deuterium scrambling in the gas phase. Finally, because the isotopic distribution for a small to medium-size peptide is essentially just the monoisotopic species ((12)C(c)(1)H(h)(14)N(n)(16)O(o)(32)S(s)), it is not necessary to deconvolve the natural abundance distribution for each partially deuterated peptide during HDX data reduction.

Electron capture dissociation of highly charged proteolytic peptides from Lys N, Lys C and Glu C digestion

Electron capture dissociation (ECD), which provides more extensive sequence coverage compared to "slow heating" tandem mass spectrometric techniques such as collision-activated dissociation, constitutes a promising method for de novo sequencing of peptides and proteins. We have previously examined and characterized the ECD fragmentation behavior of small to medium size doubly and triply protonated peptides from trypsin, chymotrypsin and Glu C digestion. Here, we extend that work to longer, more highly charged proteolytic peptides from Lys N, Lys C and Glu C digestion to compare the extent of fragmentation obtained from each type of proteolytically derived peptide and also to examine the effectiveness of each enzyme in terms of peptide sequence coverage in ECD. Our findings demonstrate that medium size peptides (i.e., 1600-4800 Da) at high charge states (+3 to +6) exhibit very similar ECD fragmentation behavior independent of which proteolytic enzyme was used for digestion. The average peptide sequence coverage we obtained for Lys N and Glu C generated peptides was 81%, and for Lys C proteolytic peptides we obtained an average sequence coverage of 80%. Most of the peptides we examined, 82%, showed high ECD sequence coverage ranging from 70-100%, whereas 13% of the peptides showed a moderate sequence coverage ranging from 50-60%. Only 5% of the peptides showed an average sequence coverage below 50%. Furthermore, the extent of fragmentation, measured by the total number of backbone c- and z-type product ions, was very similar for Lys N, Lys C and Glu C derived peptides.

Characterization of a new isolate of Lactobacillus fermentum IFO 3956 from Egyptian Ras cheese with proteolytic activity

More than 200 isolates were obtained from 15 Egyptian traditional dairy products (Domiatti cheese, Ras cheese and Rayeb milk) collected from local markets of Alexandria, Tanta and Kafr El-Sheikh. Examination with optical microscope of these dairy samples allowed to classify 92 bacilli, 64 of which were identified as lactobacilli. The proteolytic activity of lactobacilli isolates was tested on skim milk agar. Eight isolates showing a high proteolytic activity were further tested on UHT skim milk. The strain showing the highest proteolytic activity was purified and identified as Lactobacillus fermentum IFO 3656. The specific proteolytic activity of this strain and the factors affecting it (pH, temperature and presence of inhibitors) were studied. The proteolysis targeted mainly caseins (73% of whole casein), especially β-casein (85%). Smaller portions of whey proteins were proteolyzed (20%) essentially β-lactoglobulin. The proteolysis process gave rise to medium-sized peptide populations. The optimum conditions for the proteolysis activity of the studied strains were pH 6.5 and 37 °C. Proteolytic activities were very slightly affected by the increase of the temperature to 42 °C or the pH to 8.2. The protease system of Lactobacillus fermentum IFO 3956 is most probably composed from a high amount of metalloproteases and small amount of cysteine and serine proteases.

Peptide Backbone Modifications

The selective introduction or manipulation of side chains in a peptide represents a severe synthetic challenge. During the last years numerous different approaches towards the targeted modification of small to medium-sized peptides have been developed which should be presented in this review. Focusing on the manipulation of the peptide backbone, procedures concerning selective N-alkylation, C-alkylation and Cequals;O-thionation are described.

Performance Characteristics of Electron Transfer Dissociation Mass Spectrometry

We performed a large scale study of electron transfer dissociation (ETD) performance, as compared with ion trap collision-activated dissociation (CAD), for peptides ranging from approximately 1000 to 5000 Da (n approximately 4000). These data indicate relatively little overlap in peptide identifications between the two methods ( approximately 12%). ETD outperformed CAD for all charge states greater than 2; however, regardless of precursor charge a linear decrease in percent fragmentation, as a function of increasing precursor m/z, was observed with ETD fragmentation. We postulate that several precursor cation attributes, including peptide length, charge distribution, and total mass, could be relevant players. To examine these parameters unique ETD-identified peptides were sorted by length, and the ratio of amino acid residues per precursor charge (residues/charge) was calculated. We observed excellent correlation between the ratio of residues/charge and percent fragmentation. For peptides of a given residue/charge ratio, there is no correlation between peptide mass and percent fragmentation; instead we conclude that the ratio of residues/charge is the main factor in determining a successful ETD outcome. As charge density decreases so does the probability of non-covalent interactions that can bind a newly formed c/z-type ion pair. Recently we have described a supplemental activation approach (ETcaD) to convert these non-dissociative electron transfer product ions to useful c- and z-type ions. Automated implementation of such methods should remove this apparent precursor m/z ceiling. Finally, we evaluated the role of ion density (both anionic and cationic) and reaction duration for an ETD experiment. These data indicate that the best performance is achieved when the ion trap is filled to its space charge limit with anionic reagents. In this largest scale study of ETD to date, ETD continues to show great promise to propel the field of proteomics and, for small- to medium-sized peptides, is highly complementary to ion trap CAD.

IN VITRO DIGESTIBILITY OF CHINESE TARTARY BUCKWHEAT PROTEIN FRACTIONS: THE MICROSTRUCTURE AND MOLECULAR WEIGHT DISTRIBUTION OF THEIR HYDROLYSATES

Our previous study showed that in vitro pepsin digestibility of Chinese tartary buckwheat protein was relatively low compared to those of other edible seeds. In vitro pepsin digestibilities of four protein fractions of tartary buckwheat, microstructure and molecular weight (MW) distributions of hydrolysates were investigated. In vitro pepsin digestion assay showed that the digestibilities of tartary buckwheat protein fractions were albumin (81.20%), globulin (79.56%), prolamin (66.99%) and glutelin (58.09%). Scanning electron microscopy showed that albumin and globulin fractions were digested by pitting from the outer surface to the inner part and were more digestible, while prolamin and glutelin fractions resisted digestion because only the outer surfaces of their protein bodies were digested and the interior was protected. MW distribution of the hydrolysates from the four protein fractions was determined by high-performance liquid chromatography. The hydrolysates of albumin mainly consisted of polypeptides with lower MW. The hydrolysates of glutelin had larger polypeptides together with small and medium-sized peptide fractions.

Electron-transfer ion/ion reactions of doubly protonated peptides: effect of elevated bath gas temperature.

In this study, the electron-transfer dissociation (ETD) behavior of cations derived from 27 different peptides (22 of which are tryptic peptides) has been studied in a 3D quadrupole ion trap mass spectrometer. Ion/ion reactions between peptide cations and nitrobenzene anions have been examined at both room temperature and in an elevated temperature bath gas environment to form ETD product ions. From the peptides studied, the ETD sequence coverage tends to be inversely related to peptide size. At room temperature, very high sequence coverage (approximately 100%) was observed for small peptides (< or =7 amino acids). For medium-sized peptides composed of 8-11 amino acids, the average sequence coverage was 46%. Larger peptides with 14 or more amino acids yielded an average sequence coverage of 23%. Elevated-temperature ETD provided increased sequence coverage over room-temperature experiments for the peptides of greater than 7 residues, giving an average of 67% for medium-sized peptides and 63% for larger peptides. Percent ETD, a measure of the extent of electron transfer, has also been calculated for the peptides and also shows an inverse relation with peptide size. Bath gas temperature does not have a consistent effect on percent ETD, however. For the tryptic peptides, fragmentation is localized at the ends of the peptides suggesting that the distribution of charge within the peptide may play an important role in determining fragmentation sites. A triply protonated peptide has also been studied and shows behavior similar to the doubly charged peptides. These preliminary results suggest that for a given charge state there is a maximum size for which high sequence coverage is obtained and that increasing the bath gas temperature can increase this maximum.

Multivariate modelling of relationships between proteolysis and sensory quality of Präst cheese

Abstract A study was undertaken to evaluate how the proteolytic processes during cheese ripening (i.e., cleavage of native caseins, formation of medium-sized and smaller peptides and free amino acids) have an impact on the development of mouthfeel/texture and taste/aroma of maturing cheeses. Multivariate modelling of relationships between results from biochemical analyses of proteolysis and from sensory profiling of Prast samples ( N = 15 ) were done using cross-validated partial least square regressions. Differences in salt perception mainly due to different salt content were correlated with several proteolytic data. Models based on profiles of smaller peptides found by reverse-phase chromatography of ethanol (70%)-soluble fractions had a correlation with descriptors such as flavour intensity ( R = 0.83 ), off-flavour ( R = 0.86 ) and bitterness ( R = 0.72 ). Composition of free amino acid correlated with flavour intensity ( R = 0.82 ), but significant correlations for more taste-related descriptors such as off-flavour and bitterness were not found. Multivariate modelling was a useful approach to relate biochemistry of cheese ripening to cheese quality as assessed by sensory profiling.

Medium‐sized peptides as built in carriers for biologically active compounds

A growing number of oligopeptides of natural and/or synthetic origin have been described and considered as targeting structures for delivery bioactive compounds into various cell types. This review will outline the discovery of peptide sequences and the corresponding mid-sized oligopeptides with membrane translocating properties and also summarize de novo designed structures possessing similar features. Conjugates and chimera constructs derived from these sequences with covalently attached bioactive peptide, epitope, oligonucleotide, PNA, drug, reporter molecule will be reviewed. A brief note will refer to the present understanding on the uptake mechanism at the end of each section.

Identification of novel immunodominant CD4+ Th1-type T-cell peptide epitopes from herpes simplex virus glycoprotein D that confer protective immunity.

The molecular characterization of the epitope repertoire on herpes simplex virus (HSV) antigens would greatly expand our knowledge of HSV immunity and improve immune interventions against herpesvirus infections. HSV glycoprotein D (gD) is an immunodominant viral coat protein and is considered an excellent vaccine candidate antigen. By using the TEPITOPE prediction algorithm, we have identified and characterized a total of 12 regions within the HSV type 1 (HSV-1) gD bearing potential CD4(+) T-cell epitopes, each 27 to 34 amino acids in length. Immunogenicity studies of the corresponding medium-sized peptides confirmed all previously known gD epitopes and additionally revealed four new immunodominant regions (gD(49-82), gD(146-179), gD(228-257), and gD(332-358)), each containing naturally processed epitopes. These epitopes elicited potent T-cell responses in mice of diverse major histocompatibility complex backgrounds. Each of the four new immunodominant peptide epitopes generated strong CD4(+) Th1 T cells that were biologically active against HSV-1-infected bone marrow-derived dendritic cells. Importantly, immunization of H-2(d) mice with the four newly identified CD4(+) Th1 peptide epitopes but not with four CD4(+) Th2 peptide epitopes induced a robust protective immunity against lethal ocular HSV-1 challenge. These peptide epitopes may prove to be important components of an effective immunoprophylactic strategy against herpes.

The Synthesis and Study of Side‐Chain Lactam‐Bridged Peptides

AbstractFor Abstract see ChemInform Abstract in Full Text.

Zinc Complexes of a Helical 22-mer Peptide with Two Histidine Donors

A new 22-mer model peptide P22 was designed for maximal α-helicity in nonpolar media, synthesized by solid-phase methods, and isolated in an analytically pure state. It contains histidine at positions 10 and 14 representing the i, i + 4 motif which is typical for metal ion binding. Treatment with zinc halides yielded the uncharged molecular complexes (P22)ZnCl2 and (P22)ZnI2 which are the first metal complexes of medium-sized peptides to be isolated in an analytically pure state and identified by mass spectrometry. The helicity of both the free peptide and its complexes was confirmed by IR and CD spectroscopy as well as by molecular mechanics calculations. This has allowed us to propose a molecular structure which shows that the complexes are viable models for zinc-binding sections in zinc finger proteins. (© Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002)