Crude Peptide Research Articles

“On‐Resin” Disulfide Peptide Synthesis with Methyl 3‐Nitro‐2‐pyridinesulfenate

AbstractNew methodologies for the construction of full peptide structures with all disulfide bonds on the resin are attractive for the development of solid phase peptide synthesis. Detailed reaction conditions for the on‐resin disulfide bond formation have been investigated using a mild and chemically stable oxidizing reagent, methyl 3‐nitro‐2‐pyridinesulfenate (Npys‐OMe). Monocyclic oxytocin, MCH and bicyclic α‐conotoxin ImI were synthesized in both semi‐automated and full‐automated protocols. It was found that on‐resin intramolecular disulfide bond formation with Npys‐OMe proceeds with the minimal formation of peptide oligomers by adopting a solvent system with 0.4 M LiCl/DMF. Crude peptides with complete disulfide bond patterns can be obtained in high purity using both protocols. This minimized the RP‐HPLC purification step and the desired peptides were obtained with better yields. To our knowledge, this is the first fully automated construction of a bicyclic disulfide peptide on resin with more than 50 % purity in Fmoc‐based SPPS. These results suggest that Npys‐OMe is a useful reagent for the disulfide bond formation in automated protocols.

Evaluation of multifunctional activity of bioactive peptide fractions from the leaves of Nauclea diderrichii (De Wild. and T. Durand) Merrill and Ixora brachypoda DC

Bioactive peptides are known for health-promoting properties due to several pharmacological effects they modulate in physiological systems. Herein, the presence of peptides particularly cyclic peptides, was investigated in two Rubiaceae members; Nauclea diderichii (De Wild. and T. Durand) Merrill and Ixora brachypoda DC by thin layer chromatography (TLC) chemical detection, and their multifunctional bioactivity evaluated. Crude and pre-purified peptides were obtained from both plants using aqueous and solid-phase extractions, respectively. The peptide fraction of N. diderrichii showed good DPPH scavenging activity (IC50 = 3.43 µg/mL) compared to the standard Gallic acid (IC50 = 1.44 µg/mL). Peptide fractions of both plants displayed inhibitory effects (MIC = 6.25 μg/mL) against bacteria and fungi strains except Escherichia coli, relative to standard drugs (MIC = 0.31 µg/mL). Both displayed significant toxicity to brine shrimp nauplii (LC50 ≤ 220.8 μg m L−1) as well as MCF-7 and RD cancer cells (IC50 ≤ 34.0 μg m L−1). The enterovirus species B and C tested were remarkably susceptible to the peptide fractions (IC50 ≤ 3.20 μg/mL).

Proteomic Analysis of Chr 18 Proteins Using 2D Fractionation.

One of the main goals of the Chromosome-Centric Human Proteome Project (C-HPP) is detection of "missing proteins" (PE2-PE4). Using the UPS2 (Universal proteomics standard 2) set as a model to simulate the range of protein concentrations in the cell, we have previously shown that 2D fractionation enables the detection of more than 95% of UPS2 proteins in a complex biological mixture. In this study, we propose a novel experimental workflow for protein detection during the analysis of biological samples. This approach is extremely important in the context of the C-HPP and the neXt-MP50 Challenge, which can be solved by increasing the sensitivity and the coverage of the proteome encoded by a particular human chromosome. In this study, we used 2D fractionation for in-depth analysis of the proteins encoded by human chromosome 18 (Chr 18) in the HepG2 cell line. Use of 2D fractionation increased the sensitivity of the SRM SIS method by 1.3-fold (68 and 88 proteins were identified by 1D fractionation and 2D fractionation, respectively) and the shotgun MS/MS method by 2.5-fold (21 and 53 proteins encoded by Chr 18 were detected by 1D fractionation and 2D fractionation, respectively). The results of all experiments indicate that 111 proteins encoded by human Chr 18 have been identified; this list includes 42% of the Chr 18 protein-coding genes and 67% of the Chr 18 transcriptome species (Illumina RNaseq) in the HepG2 cell line obtained using a single sample. Corresponding mRNAs were not registered for 13 of the detected proteins. The combination of 2D fractionation technology with SRM SIS and shotgun mass spectrometric analysis did not achieve full coverage, i.e., identification of at least one protein product for each of the 265 protein-coding genes of the selected chromosome. To further increase the sensitivity of the method, we plan to use 5-10 crude synthetic peptides for each protein to identify the proteins and select one of the peptides based on the obtained mass spectra for the synthesis of an isotopically labeled standard for subsequent quantitative analysis. Data are available via ProteomeXchange with the identifier PXD019263.

Antibacterial Metabolites obtained from fermentation of peanut and cowpea by <i>Lactobacillus</i> spp

Lactic acid bacteria elective habitat is food matrix, where they release encrypted metabolites from several parent proteins as a result of their proteolytic activity in the matrix. These metabolites when decrypted confer different bioactive activity thus improving public health. This study was aimed at producing peptide containing metabolites with antibacterial efficacy from defatted cowpea and peanut using Lactobacillus spp. Lactobacillus spp. isolated from spoilt yoghurt were identified using culture dependent and independent method. The isolates were screened for proteolytic ability on skimmed milk agar. The selected isolate with highest proteolytic activity was used for metabolites production through fermentation of defatted cowpea and peanut at 37 oC for 72 h. Parameters analyzed during fermentation were pH, Lactobacillus count, protease activity, peptide concentration and antibacterial activity. The crude peptides produced were assayed for antibacterial activity against bacteria isolated from spoilt meat. From the identified Lactobacillus spp., L. plantarum CAU4347 had the highest proteolytic activity with clear zone of 24.50 ± 0.707 mm. During fermentation the highest and lowest Lactobacillus counts were from cowpea and peanut media with values 294.2 ± 0.21 ×107 and 0.60 ± 0.4 ×107 cfu/ml respectively. Cowpea medium inoculated with L. plantarum CAU4347, had the highest peptide concentration of 79.92 ± 0.01 µg/ml. Consequently, peanut medium showed higher antibacterial activity of 18 mm against Escherichia coli. This result finding suggests that encrypted peptide metabolites from cowpea and peanut flour can confer antibacterial activity against meat spoilage bacteria thus could be utilized as a potential bio-preservative.
 Keywords: Metabolites, Cowpea, Peanut, Bioactive, Fermentation, Lactobacillus plantarum

Effect of partial replacement of soybean meal with high-temperature fermented soybean meal in antibiotic-growth-promoter-free diets on growth performance, organ weights, serum indexes, intestinal flora and histomorphology of broiler chickens

In order to investigate the effects of the dietary supplementation of high-temperature fermented soybean meal (HFSBM) without antibiotics on broiler chickens, a total of 270 one-day-old chicks were distributed ad libitum to 5 dietary treatments with 6 pen replications of 9 birds, consisting of two control treatments (basal diet (HFSBM0, 0 g kg−1) and basal diet plus antibiotics (Ant) and three HFSBM addition treatments (50, 100 and 150 g kg−1). After fermentation with Bacillus stearothermophilus, the crude protein, peptide and total phenolic contents in HFSBM significantly increased, while trypsin inhibitor activity decreased by 74% compared to soybean meal (SBM). Growth performance was determined on day 0, 21 and 42. After the 42-d feeding experiment, one chick from each pen was picked out for sampling on day 42. Samples of blood, heart, liver, spleen, thymus, bursa of Fabricius, duodenum, jejunum, ileum and cecum were collected for analysis. Dietary supplementation with HFSBM (50, 100 and 150) presented no significant influence on growth performance of broilers, but had positive effects on increase of relative weights of thymus and bursa of Fabricius, reduction of glutamic-oxaloacetic transaminase (GOT) level in serum, improvement of intestinal morphology and gut flora. Results showed that chicks fed with HFSBM-containing diets had an increasing tendency (P = 0.111 & 0.156) on the relative weight of thymus compared to HFSBM0 and Ant controls, and also an increasing tendency (P = 0.051) on the relative weight of bursa of Fabricius compared to the Ant (days 1−42). Meanwhile, the GOT level in serum reduced tendentiously (P = 0.112) compared to HFSBM0, while decreased significantly (P = 0.024) compared to Ant. The villus height to crypt depth ratio (V/C) of the duodenum was higher (P = 0.003 & 0.008) than that in chicks fed with HFSBM0 and Ant. The count of B. subtilis in jejunum digesta increased significantly (P = 0.001 & P = 0.001) compared to two controls, and the B. subtilis count in ileum digesta enhanced significantly compared to Ant. Furthermore, the E. coli populations of digesta in duodenum and cecum decreased significantly (P = 0.001 & P < 0.001) compared with HFSBM0. The HFSBM supplementation diet is expected to be a promising antibiotic-growth-promoter-free (AGP-free) feed for broiler chickens.

Effects of Agaricus blazei polypeptide on cell senescence by regulation of Keap1/Nrf2/ARE and TLR4/NF-κBp65 signaling pathways and its mechanism in D-gal-induced NIH/3T3 cells

• Three active components ABp-RRQ, ABp-RQR and ABp-TRAR were obtained. • ABp-RQR was the main active component to alleviate the oxidative inflammatory reactions. • ABp-RQR play an anti-aging role by regulating Nrf2/ARE and TLR4/NF-κBp65 pathways. Agaricus blazei is a kind of fungus plant with an edible as well as a medicinal use. Previous experiments have shown its crude peptide to possess an obvious anti-aging effect. In the present study three active components viz . ABp-RRQ, ABp-RQR and ABp-TRAR were obtained from the Agaricus blazei polypeptide. ABp-RQR was the main active component with a molecular weight of 458 Dalton, and HPLC confirmed it’s a purity > 98%. When the D-galactose(D-gal)-induced NIH/3T3 cells were treated with ABp-RQR, changes were observed in the oxidative stress activities and the contents of inflammatory factors. Cell morphology and the percentage of positive cells were observed using the β-galactosidase staining. Western blot was used to detect the expression levels of the proteins modulated in the inflammatory and antioxidant signaling pathways. Our study suggests that ABp-RQR may play an anti-aging role by alleviating the oxidative inflammatory reactions in D-gal-induced NIH/3T3 cells.

Peptide pools for target antigen identification, immune monitoring, and cellular therapy

Background & Aim Background There is an increasing need for peptide tools in experimental and clinical immunology, particularly for personalized immunotherapies. The most important applications include target protein identification, epitope mapping/optimization, and T-cell response monitoring. Clinical-grade peptides are essential for (neo-)epitope based vaccines and immunotherapies. A multitude of available specifications can make it difficult to choose the best peptide format for a specific application. Methods, Results & Conclusion Methods Peptide pools were produced at different specifications. Purities ranged from crude (for discovery) to highly purified (for clinical applications). Crude peptide pools for antigen target discovery were produced by high-throughput peptide synthesis allowing the assembly of tens of thousands of peptides covering whole antigen families or even entire pathogens. Peptide pools for immune monitoring, by contrast, were manufactured using traditional peptide synthesis methods with added measures that avoid contamination that either impedes T-cell stimulation or causes stimulation independently. Clinical grade peptides representing tumor-associated (neo-)antigens or pathogen-derived antigens were produced to meet the highest specifications in terms of purity and absence of contaminants. Results While no peptide format is universally applicable, several tailored formats were developed that proved useful in key applications requiring T-cell stimulation. For example, unpurified but well-characterized peptide pools and individual peptides were used to measure T-cell responses against known target proteins, to discover new target proteins, and to identify stimulating peptides from a large number of candidate peptides. Only purified peptides and fully characterized pools, by contrast, provided the robust assay data needed for cell-based immune monitoring. For clinical applications, peptide and pool manufacturing were performed under line clearance in a low bioburden environment with full analytical coverage. In addition, full process traceability and documentation, as well as stability testing, were performed to fulfill regulatory requirements. Conclusions When deciding which peptide pool format to use for a specific purpose, the following aspects need to be considered: peptide length, number and amount of peptide(s), purity, avoidance/elimination of contaminations, stability, QC/QA measures, and last but not least cost.

Identification Of A Novel Histone Derived Antimicrobial Peptide In Airway Surface Liquid.

The airway surface liquid (ASL), a protective layer secreted by the airway epithelium, represents the first line of defence against inhaled infectious material. It contains a complex array of secreted proteins and peptides that aid the neutralisation and removal of inhaled microbes and toxicants. The ASL also contains many proteases that can cleave proteins to generate further bioactive peptides. We therefore hypothesised that the airway ASL sustained an extensive peptidome containing novel bioactive peptides. We examined the ASL peptide profile of normal human bronchial epithelial cells transformed with BMI‐1 (NHBE‐BMI1) and cell‐lines Calu3 (submucosal adenocarcinoma) and H441 (Clara cell like adenocarcinoma). ASL was acquired by washing the apical surface of epithelial monolayers grown at air‐liquid interface with PBS (100μl) at 0, 24 and 120 hours after exposure to hyperglycaemic (25mM glucose) or normoglycaemic (5mM glucose and 20mM mannitol) basolateral medium. The ASL peptides &lt; 10kDa, were subsequently isolated and analysed using the Q Exactive™ HF‐X Hybrid Quadrupole‐Orbitrap™ Mass Spectrometer. The resulting spectra of native peptides and their subsequent fragments were analysed using the pNovo De novo sequencing tool. Our preliminary results identified 4765 unique peptides (NHBE‐BMI1: 646, Calu3: 3150, H441: 1197). Of these, 37 peptides were common to all samples regardless of glycaemic state. A number of histone derived peptides were identified in all samples and cell lines. These peptides had a proline‐alanine rich N‐terminal region and a cationic C‐terminal, similar to antimicrobial peptides from histone H1 found in Atlantic salmon skin mucous. Synthetic peptides were produced by SPOT synthesis and the antimicrobial activity tested against luminescent Pseudomonas aeruginosa over 18 hours at 37°C in (100mM Tris (pH 7) and 20mM glucose). One of the crude peptides elicited a dose dependent inhibition of bacterial luminescence at 4.63±1.34 μM (n=3), indicating no bacteria survived. Truncated variants of this peptide containing the alanine‐proline rich N‐terminal sequence or the cationic C‐terminal sequence were produced to determine if a specific region of the peptide was required for antimicrobial activity. The N and the C terminal peptides had their antimicrobial activity significantly decreased, only reducing bacterial luminescence by 28.06±2.27% and 35.28±9.45% respectively compared to the full‐length peptide; 99.99±0.001% (all normalised to untreated bacterial luminescence, n=4, p&lt;0.0001, ±SD). This indicates that both regions are required for antimicrobial activity. An immunoblot of concentrated native ASL peptides and cell lysates showed the presence of known histone protein epitopes in our peptide of interest. Furthermore, we identified this histone‐derived peptide in sputum samples from both Cystic Fibrosis and normal healthy patients. Our preliminary data indicates that this novel histone derived peptide may serve an antimicrobial function in the ASL.Support or Funding InformationMedical Research Council London Intercollegiate Doctoral Training Programme

Advantageous use of SFC for separation of crude therapeutic peptides and peptide libraries

A simple SFC/MS (Supercritical Fluid Chromatography/Mass Spectrometry) method set was developed to effectively screen separations of various crude synthetic peptide products of pharmaceutical interest. Additives to the modifier methanol which were successful for these separations were found to include TFA (trifluoroacetic acid) and ammonia mixed with TFA, each at 0.1 % (v/v) composition in methanol. A final screening column set consisted of 2-ethylpyride (2-EP), 4-ethylpyridine (4-EP) and cross-linked diol (Luna™ HILIC) stationary phases. Small, linear and macrocyclic peptides with fewer than ten residues could all be eluted with good performance under at least one of the method conditions comprising the final screening protocol. For larger peptides either the 4-EP and HILIC columns with the above additives provided a good initial screening method set without 2-EP. The gradient was slightly longer and shifted to higher polarity relative to the gradients for smaller peptides. This method was often successful to elute large, hydrophilic peptides up to a 41-mer with acceptable peak profiles although the largest peptides with most ionizable residues were most challenging. For these peptides generally HILIC column performance was better with TFA + ammonia in the modifier than with TFA, while 4-EP performance was usually improved with TFA relative to TFA + ammonia.

Effect of crude peptide extract from mutton ham on antioxidant properties and quality of mutton patties

This study evaluated the effects of crude peptide extract (CPE) from mutton ham on the lipid and protein oxidation of mutton patties during storage. Color indices, texture, and microbiological and sensory properties were assessed. The results showed that CPE addition significantly reduced thiobarbituric acid-reactive substance levels, carbonyl content, sulfhydryl content loss, and protein cross-linking in mutton patties; moreover, CPE improved their color, texture, and microbiological stability. Sensory evaluation results showed that CPE significantly improved the color, flavor, juiciness, texture, and overall acceptability scores of the patties. Overall, CPE was suitable for the production of mutton patties with good organoleptic qualities. Practical applications In China, mutton patties are one of the most popular fast and convenient meat products. They are, however, prone to quality deterioration during storage. To the best of our knowledge, few studies have explored the effect of CPE from mutton ham on the antioxidant properties and quality of mutton patties. Herein, we report that CPE significantly inhibits the lipid and protein oxidation of mutton patties during storage, thereby improving product quality.

Evaluation of peptide-rich root extracts of Calliandria portoriscensis (Jacq.) Benth (Mimosaceae) for in vitro antimicrobial activity and brine shrimp lethality

BackgroundSeveral Host defence peptides (HDPs) are low molecular weight (< 50 amino acids residues) peptides detected in several ethnomedicinal plants and have particularly gained research interest in recent times. Due to their wide range of bioactivity, occurrence, abundance and ability to induce very little resistance, they hold promising potentials in drug development. This study investigated the presence of bioactive peptides in the roots of Calliandra portoricensis (CPr) (Mimosaceae) and evaluated its antimicrobial activity against gram-negative and gram-positive bacteria.MethodsThe crude peptide extract was obtained and pre-purified on pre-loaded tube of RP-C18 solid phase cartridges (strata giga tube C18-E; 5 g, 20 mL, Phenomenex, Germany). Peptide enriched fraction was chemically analysed for arginine-rich/aromatic amino acid-rich peptides using a modified G-250 analytical stain and ninhydrin on thin layer chromatography (TLC) for a preliminary screening. Furthermore, MALDI TOF/TOF peptidomics was used to detect the presence and masses of the peptides. Extracts from CPr were used to test the ability to inhibit microbial growth using p-INT (Para-iodonitrotetrazolium violet) dye, with 0.1% gentamycin as positive control. The concentration that inhibits the growth of microorganisms by 50% (IC50) were determined. Toxicity of the two extracts was accessed using freshly hatched nauplii of Artemia salina. Data analysis were evaluated using Microsoft excel and GraphPad Prism5.ResultsLow molecular weight (LMW) peptides were detected in CPr using TLC and MALDI-TOF MS. Generally, the extracts exhibited good inhibition (70–95%) against the gram-negative and gram-positive bacteria, except MRSA6 typed strain. Enhanced activity was observed in the pre-purified peptide fraction than in the methanol crude, except on MRSA6. The greatest antimicrobial inhibition by pre-purified peptide fraction was against MRSA22 (IC50 = 0.69 ± 0.33 μg/mL). The crude methanol extract (LC50 = 5.13 μg/mL) was slightly more toxic than the peptide extract (LC50 = 6.12 μg/mL).ConclusionsThis is the first report on detection of bioactive LMW peptides in Mimosaceae family. These peptides appear to be rich in arginine and aromatic amino acids. The peptide extract, in its pre-purified form showed a lower Brine shrimp cytotoxicity and an enhanced antimicrobial activity against the tested gram-negative and gram-positive bacteria.

Screening of Potential Bioactive Peptides from Bivalves of Donax cuneatus and Pitar erycina: A Peptide MALDI-ToF-Mass Fingerprinting Approach

The unexplored marine bivalves: Donax cuneatus and Pitar erycina are collected and the crude tissue proteins are extracted from them by diverse extraction methods. The crude protein fraction obtained from the acidic extraction method showed the highest yield and have further considered in our study. Various process parameters for the crude peptide extraction was statistically optimized based on response surface methodology revealed that the predicted peptide concentration from the polynomial model was 17.32 mg/mL, based on of the predicted process variables, i.e., 13.36 g/mL solid/liquid ratio, 26.63 °C temperature and 18.36 min of homogenization time. Subsequently, the crude peptide fraction isolated was subjected to purification by column chromatography followed by RP-HPLC. The fraction 3a from Donax cuneatus and 5b from Pitar erycina showed the highest antibacterial activity against Proteus vulgaris (20 µg/mL), Klebsiella pneumonia (10 µg/mL) and Streptococcus pyogenes (10 µg/mL) when compared to that of standard drug chloramphenicol at 40, 20 and 10 μg/mL respectively. Besides, the lowest minimum bactericidal concentration observed against Klebsiella pneumonia and Salmonella typhi for both the purified peptides was10 µg/mL. These antimicrobial peptides 3a and 5b exhibited significantly high antibacterial activity at 2× concentration of minimum inhibitory concentration, as evidenced through the in-vitro time-kill kinetic assay. They found to suppress the growth of all pathogenic bacteria under evaluation after 4 h. In addition to the above results, MALDI ToF-MS revealed the molecular mass of the peptide fractions 3a and 5b was 10.5 kDa and 10.0 kDa, respectively. Thus, the purified antimicrobial peptides isolated from the crude protein extracts from Donax cuneatus and Pitar erycina are found to be potential against the tested dreadful pathogens and could be translated for preclinical research in the future.

Antimicrobial peptides from Burkholderia arboris RAI16 inhibit diabetic wound pathogens

The occurrence of diabetic mellitus has affected millions of populations worldwide with global estimation of 6.4%, hence affecting 285 million adults in 2010. Foot ulceration and wound infection are major forms of complications prevalence to diabetic mellitus which is due to microbial infections. Approximately 20% of moderate or severe diabetic foot ulcer lead to some level of amputation. In this study, we aimed to isolate potential AMP produced by Burkholderia arboris RAI16, a bacterium previously isolated from the mucus of L. terrestris. The crude peptides of B. arboris exhibited significant antimicrobial activity against all 9 test microorganisms. However, the size of inhibition zones varied among all the microorganisms, indicating different susceptibility of the test microorganisms to the crude peptides. Among the microbial species, the largest inhibition zone was observed on P. mirabilis. Enzymatic digestion of the crude peptides revealed that activity was significantly affected by the treatment with trypsin, protease and Proteinase K, confirming the proteinaceous nature of the compound. A total of 120 fractions were collected from ion exchange chromatography. However, only three fractions (Fraction 41, 113 and 118) contain anionic peptides that exhibited significant active inhibitory activity on P. mirabilis. In conclusion, AMPs produced by B. arboris RAI16 showed significant inhibitory effect on diabetic wound microorganisms. Further investigations should be conducted to identify the isolated AMPs to provide an alternative solution for diabetic wound infections.

Limited recognition of Mycobacterium tuberculosis-infected macrophages by polyclonal CD4 and CD8 T cells from the lungs of infected mice.

Immune responses following Mycobacterium tuberculosis (Mtb) infection or vaccination are frequently assessed by measuring T-cell recognition of crude Mtb antigens, recombinant proteins, or peptide epitopes. We previously showed that not all Mtb-specific T cells recognize Mtb-infected macrophages. Thus, an important question is what proportion of T cells elicited by Mtb infection recognize Mtb-infected macrophages. We address this question by developing a modified elispot assay using viable Mtb-infected macrophages, a low multiplicity of infection and purified T cells. In C57BL/6 mice, CD4 and CD8 T cells were classically MHC restricted. Comparable frequencies of T cells that recognize Mtb-infected macrophages were determined using interferon-γ elispot and intracellular cytokine staining, and lung CD4 T cells more sensitively recognized Mtb-infected macrophages than lung CD8 T cells. Compared to the relatively high frequencies of T cells specific for antigens such as ESAT-6 and TB10.4, low frequencies of total pulmonary T cells elicited by aerosolized Mtb infection recognize Mtb-infected macrophages. Finally, we demonstrate that BCG vaccination elicits T cells that recognize Mtb-infected macrophages. We propose that the frequency of T cells that recognize infected macrophages could correlate with protective immunity and may be an alternative approach to measuring T-cell responses to Mtb antigens.

Synthesis of Antioxidant Peptide SCAP1 (Leu-Ala-Asn-Ala-Lys)

SCAP1 is a pentapeptide isolated from the hydrolysate of oyster (Saccostrea cucullata), which has antioxidant and anticancer properties. This study aims to find a novel strategy to obtain antioxidant peptide SCAP1 (Leu-Ala-Asn-Ala-Lys) through chemical synthesis. This study reports the first synthesis of SCAP1 through the solid phase peptide synthesis (SPPS) method with Fmoc strategy on 2-chlorotrityl chloride resin. A HATU-HOAt coupling reagent was employed in all amide bond formations and 95% TFA in water was employed to release the desired peptide from the solid support. The crude peptide was purified using successive preparative-RP-MPLC and RP-HPLC to obtain the pure peptide. The synthesized peptide was confirmed by mass spectrometry, showing a correct molecular ion peak at m/z 516.3143 (M+H)+, referring to the desired SCAP1. 1H-NMR spectra of the peptide revealed protons signals consistent to the structure of SCAP1. During the synthesis, the coupling between Ala and Asn(Trt) was not straightforward, which led to a low yield of the final product. The use of Trt-based Asn was not a suitable option, as it could cause aggregation during the synthesis, eventually leading to a poor yield of the final product. SCAP1 was obtained as a white solid with a percent yield of 8.28%.

The aspartimide problem persists: Fluorenylmethyloxycarbonyl‐solid‐phase peptide synthesis (Fmoc‐SPPS) chain termination due to formation of N‐terminal piperazine‐2,5‐diones

Aspartimide (Asi) formation is a notorious side reaction in peptide synthesis that is well characterized and described in literature. In this context, we observed significant amounts of chain termination in Fmoc‐SPPS while synthesizing the N‐terminal Xaa‐Asp‐Yaa motif. This termination was caused by the formation of piperazine‐2,5‐diones. We investigated this side reaction using a linear model peptide and independently synthesizing its piperazine‐2,5‐dione derivative. Nuclear magnetic resonance (NMR) data of the side product present in the crude linear peptide proves that exclusively the six‐membered ring is formed whereas the theoretically conceivable seven‐membered 1,4‐diazepine‐2,5‐dione is not found. We propose a mechanism where nucleophilic attack of the N‐terminal amino function takes place at the α‐carbon of the carbonyl group of the corresponding Asi intermediate.In addition, we systematically investigated the impact of (a) different adjacent amino acid residues, (b) backbone protection, and (c) side chain protection of flanking amino acids. The side reaction is directly related to the Asi intermediate. Hence, hindering or avoiding Asi formation reduces or completely suppresses this side reaction.

Preparation of high Fischer’s ratio corn oligopeptides using directed enzymatic hydrolysis combined with adsorption of aromatic amino acids for efficient liver injury repair

Directed hydrolysis of corn crude peptides was performed by sequential digestion with α-chymotrypsin and carboxypeptidase A. With the additional step of adsorption of aromatic amino acids by activated carbon, corn oligopeptides with a Fischer’s ratio of 41.87 and low bitterness were prepared. The fraction with molecular weight of 180–1000 Da was found to constitute 71.37% of these oligopeptides, where the proportion of Phe and Tyr among all amino acid residues was only 0.73%. In addition, a model of liver injury induced by carbon tetrachloride was established. Through the analysis of the body–mass index, liver index, liver histopathological features, and various serum biochemical indicators of liver function, high Fischer’s ratio corn oligopeptides were found to be safe and slightly superior in terms of liver injury repair when compared with a commercial high Fischer’s ratio amino acid product. Crude corn peptides (raw material) and skimmed milk powder had no effect on repairing liver injury.

Process optimization for synthesis of the anticancer drug delivery vehicle P10 peptide and its interaction with doxorubicin

In this study, we optimized the conditions for the solid-phase synthesis of P10 peptide (AAAAGGGRGD), a delivery vehicle of the anticancer drug doxorubicin (DOX), and established an optimal process for the synthesis of P10. Fluorescence spectroscopy was used to analyze the interaction between P10 and DOX. The cytotoxicity of P10 toward mouse breast cancer (4T1) cells was examined by MTT assay. The results showed that by using 2-chlorotrityl chloride resin as the carrier, N,N-diisopropylethylamine as the activation reagent, the coupling reagent TBTU as the catalyst, an amino acid-to-resin mass ratio of 0.5:1, and a reaction time of 1.5 h, the yield of crude P10 peptide could reach 87.06% with a purity of 95.55%. DOX had a good static quenching effect on the P10 fluorescence, and the intermolecular force between DOX and P10 was mainly hydrogen bonding or van der Waals force. P10 was not toxic to the 4T1 cells.

Synthetic Peptide Purification via Solid-Phase Extraction with Gradient Elution: A Simple, Economical, Fast, and Efficient Methodology

A methodology was implemented for purifying peptides in one chromatographic run via solid-phase extraction (SPE), reverse phase mode (RP), and gradient elution, obtaining high-purity products with good yields. Crude peptides were analyzed by reverse phase high performance liquid chromatography and a new mathematical model based on its retention time was developed in order to predict the percentage of organic modifier in which the peptide will elute in RP-SPE. This information was used for designing the elution program of each molecule. It was possible to purify peptides with different physicochemical properties, showing that this method is versatile and requires low solvent consumption, making it the least polluting one. Reverse phase-SPE can easily be routinely implemented. It is an alternative to enrich and purified synthetic or natural molecules.

Elevated levels of autoantibodies against EXD2 and PHAX in the sera of patients with chronic thromboembolic pulmonary hypertension

While circulating autoantibodies have been detected in patients with several cardiovascular diseases, such studies have not been performed for chronic thromboembolic pulmonary hypertension (CTEPH) and pulmonary arterial hypertension (PAH). Here we investigated the production of certain auto-antibodies in CTEPH patients. Initial screening was performed in 5 CTEPH patients and 5 healthy donors (HDs) using a ProtoArray Human Protein Microarray v5.1 containing 9,375 human proteins, and we selected 34 antigens recognized by IgG antibodies more strongly in the sera of CTEPH patients than in the sera of HDs. In subsequent second/third analyses, we validated the auto-antibody level using amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) in 96 CTEPH patients and 96 HDs as follows: At the second screening, we used 63 crude peptides derived from those selected 34 antigens and found that the serum levels of autoantibodies for 4 peptides seemed higher in CTEPH patients than in HDs. In third analysis, we used the purified peptides of those selected in second screening and found that serum antibodies against peptides derived from exonuclease 3'-5' domain-containing 2 (EXD2) and phosphorylated adaptor for RNA export (PHAX) were significantly higher in CTEPH patients than in HDs. The serum antibody levels to these antigens were also elevated in PAH patients. The titers against EXD2 peptide decreased after surgical treatment in CTEPH patients. These autoantibodies may be useful as biomarkers of CTEPH and PAH, and further investigations may provide novel insight into the etiology.