Isolation Purification and Characterization of Antimicrobial Peptides from <Emphasis Type="Italic">Cuminum cyminum</Emphasis> L. Seeds

In this work, antimicrobial peptides from Cuminum cyminum L. seeds were isolated and purified for the first time by 50% ethanol extraction, C18 reverse phase column chromatography and ion exchange chromatography for separation different peptides fraction. Then isolated fractions were characterized by Gel electrophoresis (SDS-PAGE), high-pressure liquid chromatography and the peptides components and molecular weights were determined by liquid chromatography and mass spectrometry. The extracts were tested against some strains of bacteria (E. coli and Staphylococcus aureus) and one strain of fungi (Candida albicans) using well diffusion and broth dilution assays. The extracts from C. cyminum L. seeds demonstrated a high degree of activity (some antibacterial effect) against the bacteria strains and аntifungal activity against the Candida albicans. However, the study indicates that the crude peptide extracts from C. cyminum L. seeds have promising antimicrobial and antioxidant activities that can be harnessed as leads for potential bioactive compounds.     

A Study on Immonium Ions and Immonium-Related Ions Depending on Different Collision Energies as Assessed by Q-TOF MS

Immonium ions and immonium-related ions commonly appear in the mass spectra of peptide precursor ions. An overall understanding of the variation of the abundance of these ions is beneficial for the identification of unknown peptides. Here, four peptides from mass spectrometry (MS) of sucrose phosphorylase were selected as precursor ions, and the frequency of immonium ions and immonium-related ions in a dataset containing 130 MS/MS spectra were examined. Immonium ions and immonium-related ions were mainly produced from the further fragmentation of a-, b-, and y-ions. At the optimal collision energy (CE), the immonium ions of leucine at m/z 86, isoleucine at m/z 86, glutamine at m/z 101, arginine at m/z 129, tryptophan at m/z 159, proline at m/z 70, valine at m/z 72, glutamic acid at m/z 102, phenylalanine at m/z 120, and tyrosine at m/z 136, as well as the immonium-related ions of methionine at m/z 61, lysine at m/z 84, glutamine at m/z 84, and tyrosine at m/z 91 existed in higher abundance and had higher confidence level, therefore suggesting the presence of corresponding amino acid residues well. However, the immonium ions of serine at m/z 60 and threonine at m/z 74, although showing lower abundance, were stable at high CE and had higher confidence level, indicating the presence of serine and threonine residues, respectively. The immonium ion of asparagine at m/z 87 also was a good indicator for the existence of asparagine residue.     

Characterization of Antimicrobial and Antioxidative Peptides Synthesized by <Emphasis Type="Italic">L. rhamnosus</Emphasis> C6 Fermentation of Milk

Lactobacillus rhamnosus C6 was used for milk fermentation with the aim of synthesizing antimicrobial and antioxidant peptides rich preparations. The proteolysis was checked for an incubation period of 72 h to check the extent of both bioactivities in fermentate. The 36 h incubated fermentate showed higher inhibition zone diameter against E. coli ATCC 25922 as well as antioxidant activity. Ultrafiltrate was further purified by solid phase extraction and then subjected to reverse phase chromatography. Among 12 fractions collected, higher activity containing fractions were sequenced through LC–MS and characterized. Total 49 peptide sequences identified including 13 novel sequences rich in proline with helix forming ability. Higher antimicrobial activity containing fractions have potent previously reported Casicidin-17 peptides along with a series of proline rich peptides. Antioxidant rich peptides profile contains 21 peptide of smaller sequence of mainly 9–12 amino acids with lower molecular weight. This study demonstrates the capacity of L. rhamnosus C6 to release antioxidative and antimicrobial peptide by proteolysis of milk proteins through peptide profiling and characterization.     

Characterization of a Gloverin-Like Antimicrobial Peptide Isolated from Muga Silkworm, <Emphasis Type="Italic">Antheraea assamensis</Emphasis>

Antimicrobial peptides (AMPs) are produced in all living organisms including insects in a non-specific manner, and act as innate immune defense arsenal against the invading pathogens. Muga silkworm (Antheraea assamensis) larvae were injected with Candida albicans and AMPs were isolated from the hemolymph after extracting with methanol, acetic acid and water mixture (90:1:9) and evaluated for antimicrobial activity against fungal and bacterial pathogens. Further purification was done through successive semipreparative and analytical reversed phase HPLC using C-18 column. The obtained fractions were collected, lyophilized and tested for antimicrobial activity. Among the HPLC fractions, one showed highest activity with MIC value of 64 µg/ml against Gram-negative bacteria, Escherichia coli and Enterobacter cloacae. Purity of this isolated peptide was confirmed by SDS-PAGE and TLC, and its molecular mass was determined as 9.052 kDa by MALDI-TOF mass spectrometry. From the mass fingerprinting analysis of this peptide after trypsin digestion a peptide fragment with molecular mass of 2622.7 Da was obtained. De novo sequencing of this peptide fragment following MS/MS analysis identified few amino acid residues as “KSGGGGWGS” with a total score of 46.9 with gloverin peptide of A. mylitta. The peptide inhibited biofilm formation of the Gram-negative bacterial pathogens. SEM study revealed that peptide disrupted bacterial cell wall to leach out intracellular materials and may be the major target for its antimicrobial activity.     

Purification and Characterization of Antioxidative Peptides Derived From Fermented Milk (<Emphasis Type="Italic">Lassi</Emphasis>) by Lactic Cultures

Fermentation of milk with lactic acid bacteria is the most suitable approach to enrich the bioactive peptides in fermented milk products. So in the present study, two sets of fermented milk (lassi) were prepared. The one lassi was prepared using standard Dahi culture NCDC-167(BD₄) and the other one was made with the same Dahi culture combined with Lactobacillus acidophilus NCDC-15 as an adjunct culture. The preparation steps i.e. preheat treatment and incubation period were optimized by using response surface methodology to obtain maximum antioxidant activity. Lassi prepared with adjunct culture using optimized conditions showed an antioxidant activity of 0.66?±?0.01 µM Trolox/mg protein which was significantly higher than that control (0.22?±?0.01 µM Trolox/mg protein). Out of 59 peptide fragments of β casein fermented by L. acidophilus and 24 peptides from control have been identified by LC–MS/MS. Most of the peptides showed the antioxidant activity. The therapeutic potential of fermented milk products could be improved by increased production of bioactive peptides through controlled fermentation using appropriate proteolytic starter strain.     

Characterization of Antimicrobial Peptides Isolated from the Processing by-Products of African Catfish <Emphasis Type="Italic">Clarias gariepinus</Emphasis>

Antimicrobial peptides (AMPs) as components of innate immunity system have been isolated from fish and other species. In this study, the crude proteins extracted with gradient ammonium sulfate precipitation technique from the processing by-products of African catfish Clarias gariepinus (C. gariepinus) were purified by size-exclusion chromatography and all the four obtained fractions, Clarias antimicrobial peptides I(CAP-I), CAP-II, CAP-III and CAP-IV, showed antimicrobial activity. Among of these fractions, CAP-IV showed the highest antimicrobial activity against Staphylococcus aureus, Aeromonas sobria, Aeromonas hydrophila, Escherichia coli by agar diffusion plate test and the diameter of inhibition zone was 8.34, 9.27, 6.76, 6.13 mm, respectively. The molecular weight of main peptides of CAP-IV was around 4.1 KD by SDS-PAGE analysis. CAP-IV showed antimicrobial activity against both gram-negative and gram-positive bacterial pathogens at minimum inhibitory concentrations (MICs) ranging from 105 to 420 μg/mL. The antimicrobial activity of CAP-IV was stable at wide pH range, 3–11 and was also heat-stable when temperature was below 80 °C. Freeze-thawing treatment also only had slight effects on the antimicrobial activity of CAP-IV. Besides, CAP-IV was not sensitive to the hydrolysis by pepsin and trypsin, except for protease K. These results suggest that CAP-IV isolated from C. gariepinus is potential to be developed as a new antimicrobial peptide and may partially explain the high disease resistance of African catfish C. gariepinus.     

Biochemical Characterization of HL-7 and HL-10 Peptides Identified from Scorpion Venom of <Emphasis Type="Italic">Hemiscorpius lepturus</Emphasis>

In this study, the scorpion venom of H. lepturus was fractionized by reversed phase high-pressure liquid chromatography (RP-HPLC). The sequences of two peptide fractions were identified by tandem mass spectrometry and named as HL-10 (AFPYYGHHLG) and HL-7 (YLYELAR), respectively. Antioxidant activity and cellular effect of synthetic peptides on A549 cell line were investigated. Results showed that the two peptides had high activities in radical scavenging and inhibition of lipid peroxidation in a concentration-dependent manner. The HL-10 and HL-7 peptides demonstrated cytotoxicity on A549 without any hemolytic effect. By increasing of peptide concentrations induced significantly (P?≤?0.01) activities of catalase and glutathione peroxidase. Our results showed that HL-7 peptide had higher antioxidant potency, whereas the HL-10 peptide revealed a great cytotoxicity on A549 cell line by MTT assay. Our results suggested that the peptides of H. lepturus, possessed free radicals scavenging likewise increased antioxidant enzyme activities in A549 cells.     

Chemotaxonomy of fungi in the <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> species complex performing GC/MS metabolite profiling

Hyphal anastomosis testing and molecular methods have been the primary criteria employed to understand the evolutionary and taxonomic relationships of the soil-borne fungal plant pathogen Rhizoctonia solani species complex. In this study, a metabolomics-based approach for characterizing and identifying isolates of R. solani using gas chromatography/mass spectrometry (GC/MS) metabolite profiling and footprinting was developed. Multivariate and hierarchical cluster analyses of GC/MS data provided resolution of isolates belonging to anastomosis groups (AGs) 1–6, 9, and 10 of R. solani. Clustering of R. solani AG-3 isolates, based on host origin, was also observed and attributed to metabolite-biomarkers belonging to amino, carboxylic and fatty acids. The chemotaxonomic approach using metabolomics is a high-throughput methodology that complements existing molecular approaches for the taxonomic investigation of Rhizoctonia isolates and monitoring of fungal metabolism.     

Rare triterpene glycoside of ginseng (ginsenoside malonyl-Rg<Subscript>1</Subscript>) detected in plant cell suspension culture of <Emphasis Type="Italic">Panax japonicus</Emphasis> var. <Emphasis Type="Italic">repens</Emphasis>

This paper reports for the first time about the detection and identification of ginsenoside malonyl-Rg₁ (the rare 20(S)-protopanaxatriol-type ginsenoside) in the biomass of plant cell suspension culture of Japanese ginseng (Panax japonicus C.A. Mey. var. repens). Ginsenosides were analyzed by means of high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC-ESI-MS) in positive-ion mode. Malonyl-Rg1 was identified as a result of interpretation of MS spectra obtained upon fragmentation of protonated molecular ion ([M + H]⁺) of this compound in an ionization source. Chromatographic analysis and MS spectra showed that the cells of P. japonicus var. repens cultivated in vitro contain several isomers of malonyl-Rg₁. Thus, we ascertained for the first time that, in addition to malonyl ginsenosides of 20(S)-protopanaxadiol group, the plant cell culture of ginseng P. japonicus var. repens can accumulate glycosides of 20(S)-protopanaxatriol group acylated with a malonic acid residue. The obtained results showed that, in the cells of ginseng cultivated in vitro for a long time (for 10 years and more), the assortment of secondary metabolites (ginsenosides) may be as wide as in intact plants.     

ACE Inhibitory and Antioxidant Activities of Novel Peptides from <Emphasis Type="Italic">Scorpaena notata</Emphasis> By-product Protein Hydrolysate

This study describes the isolation of angiotensin I converting enzyme and antioxidative peptides from head protein hydrolysate of red scorpionfish (Scorpaena notata) prepared by treatment with a protease from the fungus Penicillium digitatum. After ultrafiltration, three peptides were isolated by a two-step procedure: size exclusion chromatography on a Toyopearl HW-40 followed by reversed-phase high performance liquid chromatography (RP-HPLC) with a high purification yield of 2.22 mg of peptide/g of initial protein. Active peptides were then identified by nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoLC/MS–MS), corresponding to the following sequences: Gln–Gln–Pro–His–Ser–Arg–Ser–Lys–Gly–Phe–Pro–Gly–Pro (1424.724 Da), Gly–Gln–Lys–Ser–Val–Pro–Glu–Val–Arg (1000.565 Da) and Val–Glu–Gly–Lys–Ser–Pro–Asn–Val (830.448 Da). Peptides D-I, E-I and F-I showed high angiotensin-I converting enzyme inhibitory activity with an IC₅₀ values of 0.98, 1.69 and 1.44 µM, respectively as well as a synergistic antioxidant activity between the different fractions. Thus, we have demonstrated that underutilized wastes can be valorized by production of peptides that can be used as potential therapeutic compounds active against oxidative stress and hypertension.     

Isolation,Purification and Characterization of Antimicrobial Peptides Produced from <Emphasis Type="Italic">Saccharomyces boulardii</Emphasis>

Saccharomyces boulardii was used for antimicrobial peptides production. Separation process of produced antimicrobial peptides was conducted using ultrafiltration technique through dialysis membranes with porous 10 (MWCO) kDa. The inhibition activity was determined against four bacterial isolates. As a result, higher inhibition zone against Bacillus cereus were 26, 29 and 33 mm after adding 50, 75 and 100 µL of concentrated peptide, respectively. After that, peptide passed through the Sephadex G-50 column to achieve purified peptide using gel filtration. The high activity of purified peptide was confirmed based on the second peak reaching to 37 mm of bacterial inhibition zone while other peaks did not show any inhibition against tested bacteria. Some of the important characteristics of purified bioactive peptide were applied. Antimicrobial peptides stability was studied and found to be stable at pH range from 5 to 7 values studied in addition to its inhibition activity reached to 100%. Regarding thermal stability, it was observed that the peptide was fully activity at a both 60–80 °C for 30 min. Moreover, molecular weight of a peptide was identified using electrophoresis technique with SDS measured at 5792 Dalton.     

MALDI-TOF MS Supplementary database for species identification employing the yeast diversity encountered on southern Brazil grapes

The study of grape microflora is of interest when autochthonous yeasts, which are related to typical wine characteristics, are intended to be used in winemaking. The election of matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) as the first method for yeast identification was based on its accuracy and rapidity compared to alternative laboratory protocols for identification. The aims of this study are to consolidate the MALDI-TOF MS Supplementary database for environmental yeasts already constructed, to expand it through the addition of standard spectra of not included yeast species, and to discuss the grape microflora encountered in Southern Brazil. A total of 358 strains, isolated from grape berries, were submitted to protein profiling employing Biotyper and Supplementary database. Molecular biology techniques were used as alternatives to identify 6.4% of strains not promptly designated by protein profiling. These strains corresponded to the species Candida californica, Zygoascus meyerae, Candida akabanensis, Candida azyma, and Hanseniaspora vineae. The MALDI-TOF MS spectra of the identified species were added to Supplementary database. The presented results strengthen the need for further expansion of the mass spectra database to broaden its microbiological application.     

Leucocin C-607, a Novel Bacteriocin from the Multiple-Bacteriocin-Producing <Emphasis Type="Italic">Leuconostoc pseudomesenteroides</Emphasis> 607 Isolated from Persimmon

Leuconostoc pseudomesenteroides 607, isolated from persimmon fruit, was found to have high inhibitory activity against Listeria monocytogenes and several other Gram-positive bacteria. Inhibitory substances were purified from culture supernatant by ion-exchange chromatography, Sep-Pak C₁₈ cartridge, and reverse-phase high-performance liquid chromatography (RP-HPLC). Two antibacterial peptides were observed during the purification procedures. One of these peptides had a molecular size of 4623.05 Da and a partial N-terminal amino acid sequence of NH₂-KNYGNGVHxTKKGxS, in which the YGNGV motif is specific for class IIa bacteriocins. A BLAST search revealed that this bacteriocin was similar to leucocin C from Leuconostoc mesenteroides. Leucocin C-specific primers were designed and a single PCR product was amplified. Analysis of the nucleotide sequence has revealed a putative peptide differing by only one amino acid residue from the sequence of leucocin C. No identical peptide or protein has been reported in the literature, and this peptide, termed leucocin C-607, was therefore considered to be a new variant of leucocin C produced by Leuc. pseudomesenteroides 607. Another antibacterial peptide purified from the same culture supernatant had a molecular size of 3007.7 or 3121.97 Da. However, detailed information regarding this second peptide remains to be determined. Distinct characteristics, such as heat stability and inhibitory spectrum, were observed for the two bacteriocins produced by Leuc. pseudomesenteroides 607. These results suggested that Leuc. pseudomesenteroides 607 produces leucocin C-607 along with another unknown bacteriocin.     

Cationic Antimicrobial Peptides Derived from <Emphasis Type="Italic">Crocodylus siamensis</Emphasis> Leukocyte Extract,Revealing Anticancer Activity and Apoptotic Induction on Human Cervical Cancer Cells

Known antimicrobial peptides KT2 and RT2 as well as the novel RP9 derived from the leukocyte extract of the freshwater crocodile (Crocodylus siamensis) were used to evaluate the ability in killing human cervical cancer cells. RP9 in the extract was purified by a combination of anion exchange column and reversed-phase HPLC, and its sequence was analyzed by mass spectrometry. The novel peptide could inhibit Gram-negative Vibrio cholerae (clinical isolation) and Gram-positive Bacillus pumilus TISTR 905, and its MIC values were 61.2 µM. From scanning electron microscopy, the peptide was seen to affect bacterial surfaces directly. KT2 and RT2, which are designed antimicrobial peptides using the C. siamensis Leucrocin I template, as well as RP9 were chemically synthesized for investigation of anticancer activity. By Sulforhodamine B colorimetric assay, these antimicrobial peptides could inhibit both HeLa and CaSki cancer cell lines. The IC₅₀ values of KT2 and RT2 for HeLa and CaSki cells showed 28.7–53.4 and 17.3–30.8 µM, while those of RP9 were 126.2 and 168.3 µM, respectively. Additionally, the best candidate peptides KT2 and RT2 were used to determine the apoptotic induction on cancer cells by human apoptosis array assay. As a result, KT2 and RT2 were observed to induce apoptotic cell death in HeLa cells. Therefore, these results indicate that KT2 and RT2 with antimicrobial activity have a highly potent ability to kill human cervical cancer cells.     

EpiProfile Quantifies Histone Peptides With Modifications by Extracting Retention Time and Intensity in High-resolution Mass Spectra

Histone post-translational modifications contribute to chromatin function through their chemical properties which influence chromatin structure and their ability to recruit chromatin interacting proteins. Nanoflow liquid chromatography coupled with high resolution tandem mass spectrometry (nanoLC-MS/MS) has emerged as the most suitable technology for global histone modification analysis because of the high sensitivity and the high mass accuracy of this approach that provides confident identification. However, analysis of histones with this method is even more challenging because of the large number and variety of isobaric histone peptides and the high dynamic range of histone peptide abundances. Here, we introduce EpiProfile, a software tool that discriminates isobaric histone peptides using the distinguishing fragment ions in their tandem mass spectra and extracts the chromatographic area under the curve using previous knowledge about peptide retention time. The accuracy of EpiProfile was evaluated by analysis of mixtures containing different ratios of synthetic histone peptides. In addition to label-free quantification of histone peptides, EpiProfile is flexible and can quantify different types of isotopically labeled histone peptides. EpiProfile is unique in generating layouts (i.e. relative retention time) of histone peptides when compared with manual quantification of the data and other programs (such as Skyline), filling the need of an automatic and freely available tool to quantify labeled and non-labeled modified histone peptides. In summary, EpiProfile is a valuable nanoflow liquid chromatography coupled with high resolution tandem mass spectrometry-based quantification tool for histone peptides, which can also be adapted to analyze nonhistone protein samples.The nucleosome, the basic unit of chromatin, consists of 147 base pairs of DNA wrapped around histone proteins (H2A, H2B, H3, and H4). Histones play vital roles in chromatin, interacting with many signaling proteins and chromatin-structural proteins through various post-translational modifications (PTMs)¹ (1–3). There are numerous PTMs on histones, including methylation (mono - me1, di - me2, tri - me3), acetylation (ac), phosphorylation (ph), ubiquitination, and SUMOylation (4). Histone PTMs can affect chromatin function, and therefore influence processes such as gene accessibility, DNA repair and chromosome condensation. Moreover, histone PTMs cross-talk in a synergistic manner to fine-tune gene expression (5). Therefore, quantification of histone PTMs has become a high priority to investigate cell regulation and epigenetics (6).Traditionally, antibody-based methods (e.g. Western blot) have been used to analyze histone modifications (7), which have multiple disadvantages. First, antibodies are not available for every new PTM discovered. Second, PTMs on neighboring amino acids (e.g. H3K9me1–3 and H3S10ph) may prevent antibody binding, a phenomenon called epitope occlusion. Third, the quantification of PTMs via antibody-based methods is not sensitive to small differences (e.g. <twofold). Mass spectrometry (MS) has emerged as a sensitive and efficient technique to detect known and novel PTMs (8). The high mass accuracy and the high speed of modern mass spectrometers allow for sensitive, confident, and accurate peptide quantification when coupled with nanoflow liquid chromatography (nanoLC).NanoLC-MS/MS analysis of protein digests (i.e. bottom-up MS) is nowadays a mature and widely applied technology. Data-dependent acquisition is the most commonly adopted MS acquisition method to identify peptides via bottom-up MS (9–12), generating MS1 and MS2 spectra. Nevertheless, histone proteins are particularly challenging to analyze by using the generalized bottom-up workflow. As histones are rich with lysines and arginines, tryptic digest of histones generates short peptides that are difficult to be retained on C18 columns. To improve histone peptide retention, the unmodified and mono-methylated lysines and peptide N terminus can be selectively chemically propionylated (13–16), preventing tryptic digest after lysine to generate longer peptides. Moreover, peptide identification through traditional database searches leads to a large number of false positives, as allowing several dynamic modifications (e.g. me1/me2/me3, ac, ph) dramatically increases the number of molecular candidates and thus the possibility to achieve a false hit (12). Therefore, software tools that quantify histone peptides require additional data to correctly map a given peptide, such as previous knowledge of peptide retention time.Quantification of histone peptides is particularly challenging because of presence of isobaric peptides, near isobaric PTMs such as tri-methylation (42.047 Da) and acetylation (42.011 Da), and low abundant species. Previous knowledge about relative peptide retention time (RT) enables differentiation between species close in mass and therefore selection of the correct peak for integration of the area of the chromatographic peak (i.e. area under curve or AUC). However, determination of peptide RT might be difficult because of their low abundance though acid extraction was performed to purify histones. This problem can be solved by using isotopically labeled synthetic histone peptides (17), or data independent approaches (18). When using relative retention time information to assign peak identities, reproducible nanoLC is crucial, especially because some isobaric peptides co-elute. In this case, the MS acquisition method must perform targeted MS2 for the co-eluting isobaric peptides at the specific time that they elute. These species can be discriminated and quantified based on the intensity of fragment ions unique to each species. For instance, the peptides KacSTGGKAPR (H3K9ac) and KSTGGKacAPR (H3K14ac) have the same mass and overlap at the nanoLC elution (the full protein sequence of human canonical histone H3 and H4 are shown in Fig. 1A). Thus, the co-eluting isobaric peptides could not be quantified separately based on the MS1 signal, but the unique fragment ions present in MS2 spectra allow them to be quantified individually.Open in a separate windowFig. 1.Histones are a challenge for quantitative mass spectrometry analyses. A, Human histone H3.1 and H4 protein sequences. B, Spline fitting to calculate AUC: blue lines are the original peaks and pink lines are the fitted peaks. C, An example of isobaric PTM modified peptides. The above MS2 is matched with H3K18ac, and the same MS2 is also matched with H3K23ac below. D, The workflow of EpiProfile: inputting precursor m/z and charge state, extracting elution profiles, selecting the correct chromatographic peak, calculating AUC, and outputting quantification tables and figures.There have been few computational investigations attempting to solve the problem of quantifying co-eluting isobaric peptides. DiMaggio et al. used a mixed integer linear optimization (MILP) framework to quantify partially co-eluting isobaric histone peptides from electron transfer dissociation (ETD) spectra (19). The framework is comprised of two MILP models: (1) enumerating the entire space of the modified forms that satisfy a given peptide mass and (2) determining the relative composition of the modified forms in the spectrum. Another study by Guan et al. identified isobaric peptides by searching ETD MS/MS spectra for ions representing all possible configurations of modified peptides using a visual assistance program. The relative abundances of these species were estimated by using a nonnegative least squares procedure (20). Other quantification programs can also perform accurate peak picking, but are commonly not as suitable for heavily modified and isobaric histone peptides (e.g. Skyline) (21). These software programs are unable to provide the layouts of histone peptides (i.e. relative RTs) or discriminate all isobaric modified peptides, two tasks that are vital for full characterization of a histone sample.In this study, we developed a new quantification program named EpiProfile. EpiProfile extracts ion chromatography for known histone peptides by using previous knowledge about their elution profiles. Moreover, it discriminates and quantifies the isobaric histone peptides by resolving the linear equations listed with the peak heights of unique fragment ions between the two modification sites in the MS2 spectra (e.g. ions between H3K9ac and H3K14ac). We evaluated the accuracy of EpiProfile by mixing different ratios of synthetic histone peptides, and then tested EpiProfile by analyzing nanoLC-MS/MS data sets of the following samples: purified histones from HeLa cells, a synthetic histone peptide library, and histone peptides labeled during cell growth with ¹³C-labeled glucose media or stable isotope labeling by amino acids in cell culture (SILAC) (22). We compared EpiProfile to manual quantification of the data, and also with the openly available program Skyline. We found that manual quantification is obviously time-consuming and that Skyline cannot generate the layouts of histone peptides and cannot discriminate four or six-component isobaric peptides, a common occurrence in histone data. Moreover, EpiProfile is highly flexible, and thus it can be used to analyze various protein samples, including isotopically labeled peptides and nonhistone data sets.     

Synthetic Antimicrobial Peptides: I. Antimicrobial Activity of Amphiphilic and Nonamphiphilic Cationic Peptides

Comparative antimicrobial properties of three artificial cationic synthetic antimicrobial peptides (SAMP): (RAhaR)₄AhaβA (where R is Arg, Aha is 6-aminohexanoic acid, βA is beta-alanine), (KFF)₃K and R₉F₂ with various amphiphilic properties have been studied relative to pathogenic strains of microorganisms: Gram-negative bacteria Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis, and Salmonella enterica, Gram-positive bacteria Staphylococcus aureus, and pathogenic yeast fungus Candida albicans. The selectivity index (SI) values of the peptide preparations were calculated as the ratio of the 50% cytotoxic concentration (TC₅₀) towards eukaryotic host cells to the MIC₅₀ values of the testing antimicrobial peptides. The studied SAMPs appeared to be the most active against the pathogenic yeast fungus C. albicans and the bacterial strains St. aureus and P. aeruginosa. The SI values in these cases exceed 40. Some assumed molecular interactions of the studied SAMPs on the microbial cells have been considered, and possible pathways to increase their antimicrobial activity have been suggested. The proposed SAMPs can serve as a basis for the design and synthesis of new promising synthetic antimicrobial agents.     

Antimicrobial activity of endogenous peptides of the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Plant and animal cells contain pools of endogenous peptides, which are the degradation products of functionally active proteins. It is known that these peptides can possess biological activity; however, the functions of most of them are unknown. The goal of the present study was to estimate the antimicrobial potential of endogenous peptides resulting from the degradation of functional proteins in cells of the moss Physcomitrella patens. Earlier, 117 peptides possessing an antimicrobial potential predicted in silico have been identified in the peptidomes of three types of P. patens cells by mass spectrometry. In the present work, the antimicrobial activity of six of these peptides toward the gram-positive bacteria Bacillus subtilis SHgw and Clavibacter michiganensis pv. michiganensis and gram-negative bacteria Escherichia coli K12 and Xanthomonas arboricola 3004 has been revealed. The results have shown that three of six peptides inhibit the growth of the phytopathogenic bacteria X. arboricola and C. m. pv. michiganensis; four peptides inhibit the growth of the gram-negative bacterium E. coli K12, and one peptide inhibits the growth of the gram-positive bacterium B. subtilis. It has been found that the peptides inhibiting the bacterial growth are predominantly the fragments of ribosomal proteins. The work confirms the potential of the biological activity of peptides that are the degradation products of functional proteins.     

Comparative muscle proteomics/phosphoproteomics analysis provides new insight for the biosafety evaluation of <Emphasis Type="Italic">fat</Emphasis>-<Emphasis Type="Italic">1</Emphasis> transgenic cattle

The biosafety of fat-1 transgenic cattle has been a focus of our studies since the first fat-1 transgenic cow was born. In this study, we used tandem mass tag labeling, TiO₂ enrichment, and nanoscale liquid chromatography coupled with tandem mass spectrometry (nanol LC–MS/MS) to compare proteomic and phosphoproteomic profiling analyses of muscle between fat-1 transgenic cows and wild-type cows. A total of 1555 proteins and 900 phosphorylation sites in 159 phosphoproteins were identified in the profiling assessments, but only four differentially expressed proteins and nine differentially expressed phosphopeptides were detected in fat-1 transgenic cows relative to wild-type cows. Bioinformatics analyses showed that all of the identified proteins and phosphoproteins were mainly related to the metabolic processes of three major nutrients: carbohydrates, lipids, and proteins. All of these differentially expressed proteins might take part in DNA recombination, repair, and regulation of the immune system. In conclusion, most of the identified proteins and phosphoproteins exhibited few changes. Our results provide new insights into the biosafety of fat-1 transgenic cattle.     

Acaricidal and repellent effects of <Emphasis Type="Italic">Cnidium officinale</Emphasis>-derived material against <Emphasis Type="Italic">Dermanyssus gallinae</Emphasis> (Acari: Dermanyssidae)

The acaricidal activity of a methanolic extract and fractions from the rhizome of Cnidium officinale against Dermanyssus gallinae adults was investigated. The C. officinale methanolic extract exhibited 100% acaricidal activity after 48 h of treatment at a dose of 4000 ppm. The acaricidal constituents of the plant were sequentially partitioned with several solvents and then purified using silica gel column chromatography and high-performance liquid chromatography. Gas chromatography–mass spectrometry and nuclear magnetic resonance spectroscopy revealed (Z)-ligustilide as a constituent of C. officinale. Acaricidal activity was examined in three experimental tests (spray, fumigation and contact), with the spraying method being the most effective. The methanolic extract of C. officinale showed both contact and fumigant activities, though only fumigant activity was observed with (Z)-ligustilide. The fumigant effects of the methanolic extract and (Z)-ligustilide caused 86.5 and 62.6% mortality, respectively, of D. gallinae adults at 48 h. Among (Z)-ligustilide, acaricides (bifenthrin, cypermethrin and spinosad) and butylidenephthalide, bifenthrin displayed the highest acaricidal activity, and the activity of butylidenephthalide was 2.3-fold higher than that of (Z)-ligustilide. These results suggest that C. officinale-derived material can be used for the development of a control agent for D. gallinae.