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1.
Upon hydrolysis with chymotrypsin, soy glycinin has a strong tendency to aggregate. The regions of glycinin from which the aggregating peptides originate were identified by accumulative-quantitative peptide mapping. To this end, the aggregating peptides were further hydrolyzed with trypsin to obtain peptides of which the sequence can be identified using RP-HPLC-MS/MS. This resulted in a hydrolysate in which 90% of the proteinaceous material was dissolved. The soluble fraction was analyzed using the method of accumulative-quantitative peptide mapping: fractionation using ion exchange chromatography, followed by identification of peptides by RP-HPLC-MS/MS, quantification based on the absorbance at 214 nm, and finally peptide mapping. For the peptide mapping the proportions in which each of the five glycinin subunits are present, as determined by Edman degradation, were taken into account. The results showed that mainly the basic polypeptide and a part of the acidic polypeptide, close to the location of the disulfide bridge between the basic and acidic polypeptides, are present in the aggregating peptide fraction. On the basis of the results obtained, an aggregation mechanism was proposed. The hydrophilic acidic polypeptides shield the hydrophobic basic polypeptides, and the former are preferentially degraded upon hydrolysis. This results in a net increase in hydrophobicity of the remaining material, which mainly consists of the basic polypeptide fragments. This increase in hydrophobicity is proposed to be the driving force in the aggregation of chymotrypsin-derived peptides of glycinin.  相似文献   

2.
The molar extinction coefficients of 20 amino acids and the peptide bond were measured at 214 nm in the presence of acetonitrile and formic acid to enable quantitative comparison of peptides eluting from reversed-phase high-performance liquid chromatography, once identified with mass spectrometry (RP-HPLC-MS). The peptide bond has a molar extinction coefficient of 923 M(-1) cm(-1). Tryptophan has a molar extinction coefficient that is approximately 30 times higher than that of the peptide bond, whereas the molar extinction coefficients of phenylalanine, tyrosine, and histidine are approximately six times higher than that of the peptide bond. Proline, as an individual amino acid, has a negligible molar extinction coefficient. However, when present in the peptide chain (except at the N terminus), it absorbs approximately three times more than a peptide bond. Methionine has a similar molar extinction coefficient as the peptide bond, while all other amino acids have much lower molar extinction coefficients. The predictability of the molar extinction coefficients of proteins and peptides, calculated by the amino acid composition and the number of peptide bonds present, was validated using several proteins and peptides. Most of the measured and calculated molar extinction coefficients were in good agreement, which shows that it is possible to compare peptides analyzed by RP-HPLC-MS in a quantitative way. This method enables a quantitative analysis of all peptides present in hydrolysates once identified with RP-HPLC-MS.  相似文献   

3.
Angiotensin I-converting enzyme (ACE), a dipeptidyl carboxypeptidase, catalyzes the conversion of Angiotensin I to the potent vasoconstrictor Angiotensin II and plays an important physiological role in regulating blood pressure. Inhibitors of angiotensin 1-converting enzyme derived from food proteins are utilized for pharmaceuticals and physiologically functional foods. ACE inhibitory properties of different enzymatic hydrolysates of glycinin, the major storage protein of soybean, have been demonstrated. The IC50 value for the different enzyme digests ranges from 4.5 to 35 microg of N2. The Protease P hydrolysate contained the most potent suite of ACE inhibitory peptides. The ACE inhibitory activity of the Protease P hydrolysate after fractionation by RP-HPLC and ion-pair chromatography was ascribed to a single peptide. The peptide was homogeneous as evidenced by MALDI-TOF and identified to be a pentapeptide. The sequence was Val-Leu-Ile-Val-Pro. This peptide was synthesized using solid-phase FMOC chemistry. The IC50 for ACE inhibition was 1.69 +/- 0.17 microM. The synthetic peptide was a potent competitive inhibitor of ACE with a Ki of 4.5 +/- 0.25 x 10(-6) M. This peptide was resistant to digestion by proteases of the gastrointestinal tract. The antihypertensive property of this peptide derived from glycinin might find importance in the development of therapeutic functional foods.  相似文献   

4.
For the isolation and detection of food-derived peptides in blood, an approach based on the derivatization of peptides with phenyl isothiocyanate (PITC) was developed. This approach allows hydrophilic peptides to be resolved and specifically detected by reversed-phase (RP) HPLC. For the rapid capturing and clarification of peptides in human plasma, solid-phase extraction by using a mini spin column (5 mmx5 mm) packed with a strong cation exchanger was used. The clarified peptide fraction was further fractionated by size-exclusion chromatography (SEC). The peptides in the SEC fractions were derivatized with PITC, and the derivatives were resolved by RP-HPLC by using an ammonium acetate buffer or a trifluoroacetic acid system. An automatic peptide sequencer based on Edman degradation with a modified program can directly analyze the resolved derivatives. Some synthetic peptides and food-derived peptides in human plasma were successfully isolated and identified by this approach.  相似文献   

5.
Somatotropins, which are used in cattle for growth and lactating performances, are difficult to reliably detect because no direct method exists. Reversed-phase high-performance liquid chromatography (RP-HLC) coupled to electrospray ionization quadrupole mass spectrometry (ESI/MS) has been developed to separate and characterize the N-terminal peptides resulting from tryptic cleavage of natural and recombinant growth hormones from different species (bovine, porcine, and human) and suppliers. Conditions for tryptic digestion were optimized. This technique was found to be optimal to cleave efficiently the N-terminal peptide of the proteins without releasing too much noise from the matrix. Characterization of the peptides through ESI(+)-MS allowed natural and recombinant growth hormones from bovine and porcine species with N-terminal amino acid sequences differing from one amino acid residue to be discriminated. However, the studied human growth hormones had similar primary sequences that did not permit any discrimination between recombinant and natural forms, thus confirming the known identity of these hormones. Protein digestions with pepsin and chymotrypsin were also compared but were not conclusive due to the too small N-terminal peptides released after proteolysis.  相似文献   

6.
Enzymatic digestion of total protein along with liquid chromatography/tandem mass spectrometry (LC/MS/MS) was used to confirm the presence of a major peanut allergen in food. Several peptides obtained from the enzymatic digestion of the most abundant peanut allergen, Ara h 1, were identified as specific peptide biomarkers for peanut protein. Using ice cream as a model food matrix, a method was developed for the detection of the allergen peptide biomarkers. A key component of the method was the use of molecular mass cutoff filters to enrich the Ara h 1 in the protein extracts. By applying the method to ice cream samples containing various levels of peanut protein, levels as low as 10 mg/kg of Ara h 1 could routinely be detected. This method provides an unambiguous means of confirming the presence of the peanut allergen, Ara h 1, in foods and can easily be modified to detect other food allergens.  相似文献   

7.
鸵鸟异嗜白细胞抗菌肽的分离纯化及部分性质研究   总被引：1，自引：0，他引：1  
目的：分离纯化出鸵鸟异嗜白细胞抗菌肽,并对其部分特性进行研究,为开发新一代高效肽类抗菌药提供依据。方法：使用氯化铵裂解、超声波破碎、醋酸提取、CM-Sepharose F F弱酸性阳离子交换层析和反相高效液相色谱（RP-HPLC）等方法,从鸵鸟异嗜白细胞中分离纯化出了抗菌肽,使用微量琼脂糖弥散法进行了抗菌活性与最小抑菌浓度（MIC）的测定,应用二级质谱（MS/MS）对抗菌肽的分子量进行测定。结果：在鸵鸟异嗜白细胞中存在抗菌肽,其对金黄色葡萄球菌S.aureus 1056MRSA、鸡大肠杆菌E.coli O78和白色念珠菌C.albicans ATCC10231具有较强的抑制作用;抗菌肽具有阳离子性质;热稳定性良好;经RP-HPLC纯化得到峰6、峰16和峰24,峰6对白色念珠菌的MIC为0.065μg/mL,峰16的分子量为4012.472Da,对鸡大肠杆菌和金黄色葡萄球菌的MIC分别为3.971μg/mL和5.245μg/mL,峰24的分子量为3542.479Da,对鸡大肠杆菌和金黄色葡萄球菌的MIC分别为26.472μg/mL和21.561μg/mL。结论：本研究得到的抗菌肽与已报道的鸵鸟抗菌肽ostricacins相比显示出更强的抑菌活性,是否为同一物质需要进一步的验证。  相似文献   

8.
为了实现竹节虾加工副产物的高值化利用,以竹节虾加工废弃物中的虾头副产物为原料,以水解度和DPPH清除率作为评价指标,采用中性蛋白酶酶解,通过响应面法优化超声辅助酶解工艺,并依次通过超滤、凝胶层析色谱和反相高效液相色谱等分离方法,从竹节虾虾头酶解产物中分离制备抗氧化肽,采用超高压液相色谱串联质谱联用技术对肽的结构进行表征。结果表明,在中性蛋白酶添加量为3 000 U·g~(-1)、p H值7.0条件下,最佳超声辅助酶解工艺参数为超声时间41 min,超声温度55℃,超声频率22 k Hz,料液比1∶9(w/v),在此条件下获得的酶解产物DPPH清除率达69.50%。当水解时间为0.5~2.5 h时,超声辅助酶解的酶解产物水解度和DPPH清除率比非超声辅助酶解工艺分别高17.95%和18.83%,该工艺缩短了酶解时间,节约了能耗。酶解产物经超滤初步分离发现,相对分子质量在3k Da(SHP4)的组分具有显著的抗氧化活性。用凝胶层析法进一步分离纯化SHP4组分后得到4个峰,其中SHP4-II的DPPH清除率最高;SHP4-II通过反相高效液相纯化后也得到4个主要肽峰,在多肽含量为1.5 mg·m L~(-1)时,SHP4-II-4的DPPH清除率最高,达到85.69%,并且具有较好的分离度,质谱分析发现,该抗氧化肽的结构为Gly-Asn-Gly-Leu-Pro(455.99 Da)。本研究结果为虾肽抗氧化保健食品的研发提供了一定的科学依据。  相似文献   

9.
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of the tryptic digest of a cleaned-up food matrix extract was used for the detection of milk allergens. The emphasis of this study was on casein, which is the most abundant milk protein and is also considered the most allergenic. A sample cleanup method was developed using an ion exchange column and centriprep device. Cookies spiked with milk powder from 0 to 1250 ppm were extracted, cleaned up, and either digested directly by trypsin or further cleaned up by gel electrophoresis before digestion. The peptide mixture was analyzed on a capillary LC-quadrupole time-of-flight system. Two marker peptides from alphaS1-casein were identified and used for prescreening. The MS/MS data from the mass spectrometry system were processed with Masslynx v4.0 and submitted for database search using either ProteinLynx Global Server or Mascot for protein identification. The LC-MS/MS method, using casein enzyme-linked immunosorbent assay as a reference, was tested on the cookie matrix and was extended to other sample matrices. There were good agreements between the two. This LC-MS/MS method provides a valuable confirmatory method for the presence of casein. It also allows the simultaneous detection of other milk allergens.  相似文献   

10.
Among tree nut allergens, pecan allergens remain to be identified and characterized. The objective was to demonstrate the IgE-binding ability of pecan 11S legumin and characterize its sequential IgE-binding epitopes. The 11S legumin gene was amplified from a pecan cDNA library and expressed as a fusion protein in Escherichia coli. The native 11S legumin in pecan extract was identified by mass spectrometry/mass spectrometry (MS/MS). Sequential epitopes were determined by probing the overlapping peptides with three serum pools prepared from different patients' sera. A three-dimensional model was generated using almond legumin as a template and compared with known sequential epitopes on other allergenic tree nut homologues. Of 28 patients tested by dot blot, 16 (57%) bound to 11S legumin, designated Car i 4. MS/MS sequencing of native 11S legumin identified 33 kDa acidic and 20-22 kDa basic subunits. Both pecan and walnut seed protein extracts inhibited IgE binding to recombinant Car i 4, suggesting cross-reactivity with Jug r 4. Sequential epitope mapping results of Car i 4 revealed weak, moderate, and strong reactivity of serum pools against 10, 5, and 4 peptides, respectively. Seven peptides were recognized by all three serum pools, of which two were strongly reactive. The strongly reactive peptides were located in three discrete regions of the Car i 4 acidic subunit sequence (residues 118-132, 208-219, and 238-249). Homology modeling of Car i 4 revealed significant overlapping regions shared in common with other tree nut legumins.  相似文献   

11.
beta-Lactoglobulin (betaLg) was hydrolyzed by plasmin to a degree of hydrolysis of 4%. The hydrolysate was fractionated by ion-exchange chromatography and subsequent hydrophobic-interaction chromatography. The betaLg peptide fraction consisting of smaller peptides (mostly <2 kDa) had poor foam- and emulsion-forming and -stabilizing properties. Most of the betaLg peptides were identified (in either the nonreduced or reduced form) by mass spectrometry on the basis of the known primary structure of the intact protein and the specificity of the enzyme. The peptides formed during betaLg/plasmin-hydrolysis were (1) peptides lacking a cysteyl residue, (2) peptides composed of a single amino acid chain containing intramolecular disulfide bonds, and (3) peptides composed of two amino acid chains linked by an intermolecular disulfide bond. It appeared that significant SH/SS-exchange had taken place during hydrolysis. Many of the peptides present in the peptide fraction that exhibited good functional properties were disulfide-linked fragments.  相似文献   

12.
The antioxidant activity of casein calcium peptides in several in vitro assay systems was investigated. Casein calcium peptides were prepared by the microbial enzymic hydrolysis of casein calcium. The main peak of the molecular mass distribution of the peptides was about 3 kDa. Casein calcium peptides showed strong antioxidant activity with the beta-carotene bleaching method, and they also showed scavenging activity against radicals such as superoxide radicals, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, and hydroxyl radicals. Antioxidant activity was increased with an increasing peptide concentration. Casein calcium peptides also showed strong antioxidant activity against lipid oxidation in ground beef homogenates. These results suggest that casein calcium peptides are a suitable natural antioxidant that prevents the lipid oxidation of meat and related food ingredients.  相似文献   

13.
Beta-lactoglobulin (beta-Lg) was subjected to high pressures up to 400 MPa and proteolysis with chymotrypsin. The hydrolysates were analyzed by SDS-PAGE and RP-HPLC, and the fragments obtained were identified by ESI-MS/MS. The results obtained showed that beta-Lg was hydrolyzed by chymotrypsin in a "progressive proteolysis" manner at either atmospheric or high pressure. Proteolysis during or after high-pressure treatment showed longer and more hydrophobic peptides than proteolysis at atmospheric pressure. Chymotrypsin showed a behavior similar to that of trypsin, with some differences, probably related to the orientation of the target residues specific for each enzyme. The similarities between proteolytic fragments produced by the two enzymes support that proteolysis enhancement under high pressure depends on the substrate changes rather than the enzyme. High pressure seemed to accelerate the first steps of proteolysis, probably through dimer dissociation, while leaving portions of the molecule more resistant to the enzyme.  相似文献   

14.
Although many sequences and linear IgE epitopes of allergenic proteins have been identified and archived in databases, structural and physicochemical discriminators that define their specific properties are lacking. Current bioinformatics tools for predicting the potential allergenicity of a novel protein use methods that were not designed to compare peptides. Novel tools to determine the quantitative sequence and three-dimensional (3D) relationships between IgE epitopes of major allergens from peanut and other foods have been implemented in the Structural Database of Allergenic Proteins (SDAP; http://fermi.utmb.edu/SDAP/). These peptide comparison tools are based on five-dimensional physicochemical property (PCP) vectors. Sequences from SDAP proteins similar in their physicochemical properties to known epitopes of Ara h 1 and Ara h 2 were identified by calculating property distance (PD) values. A 3D model of Ara h 1 was generated to visualize the 3D structure and surface exposure of the epitope regions and peptides with a low PD value to them. Many sequences similar to the known epitopes were identified in related nut allergens, and others were within the sequences of Ara h 1 and Ara h 2. Some of the sequences with low PD values correspond to other known epitopes. Regions with low PD values to one another in Ara h 1 had similar predicted structure, on opposite sides of the internal dimer axis. The PD scale detected epitope pairs that are similar in structure and/or reactivity with patient IgE. The high immunogenicity and IgE reactivity of peanut allergen proteins might be due to the proteins' arrays of similar antigenic regions on opposite sides of a single protein structure.  相似文献   

15.
Analysis by SDS-PAGE and GPC-MS of fermented cocoa extracts shows changes in the amount and composition of the major proteins, accompanied by formation of complex distributions of peptides. MS/MS studies and application of SEQUEST sequencing software have allowed identification of two related peptides, a hexapeptide and a nonapeptide, formed from vicilin, one of the cocoa storage proteins. Time course studies of the two peptides show different abundance profiles and indicate, in part, production of the hexapeptide from the nonapeptide.  相似文献   

16.
Chromatographic fractionation of crude extracts (C8 extracts) from the protein-enriched flour of commercial field peas (Pisum sativum L.) has been shown here to yield peptide mixtures related to the pea albumin 1b (PA1b) family of cysteine-rich plant peptides. The mixtures were obtained initially by flash chromatography with silica gel. Following elution of soyasaponins and lysolecithins, the end fractions obtained with the use of two flash chromatographic solvent systems displayed activity in a flour disk antifeedant bioassay with the rice weevil [Sitophilus oryzae (L.)]. Chemical properties of these mixtures were compared by thin-layer chromatography, high-performance liquid chromatography (HPLC), IR, MS, and amino acid analyses. The major peptides of C8 extracts, with average masses of 3752, 3757, and 3805 Da, were isolated by anion exchange chromatography. Samples enriched in the peptide of mass 3752 were isolated by cation exchange chromatography. Reduction plus alkylation experiments in combination with electrospray ionization mass spectrometry showed that C8 extracts contained about 10 peptides and, like PA1b, each peptide possessed six cysteine residues (three disulfide bonds). Disulfide bond reduction with 2-mercaptoethanol destroyed the antifeedant activity. The native peptides of C8 extracts were found to be resolved into nine peaks with XTerra HPLC columns operating at alkaline pH. These columns were employed to assess the distribution of pea peptides in the isolated fractions, with photodiode array and electrospray detection.  相似文献   

17.
Purification of lactoferricin (Lfcin), a cationic antimicrobial peptide, was achieved by peptic digestion of food grade bovine lactoferrin (LF) followed by fractionation on an industrial grade cation exchange resin with stepwise salt gradient elution. The digest and eluted fractions were partially characterized by MALDI-ToF MS and N-terminal sequencing. A fraction eluted using phosphate buffer with 2.0 M NaCl contained predominantly two peptides with masses of 3196 and 3124 Da, which corresponded to the 26- and 25-amino acid peptides FKCRR WQWRM KKLGA PSITC VRRAF (A), containing the Lfcin sequence. Putative sequences of cationic peptides in other eluted fractions included FKNKS RSFQ, WRMKK LGAPS ITCVR RA, and GAPSI TCVRR AFALE CIRAI AEKKA. The iron saturation level of LF had no effect on the production of Lfcin. Nevertheless, the digestion of LF containing lower iron content led to the production of a higher quantity of low molecular weight cationic peptides. A two-step process using industrial grade cation exchange resin led to 35% recovery of Lfcin and also produced other cationic peptides with potential bioactive properties.  相似文献   

18.
An MS-based method, combining reversed-phase capillary liquid chromatography (capillary LC) with quadrupole time-of-flight tandem mass spectrometry (nano-ESI Q-TOF MS/MS), was developed with the aim of identifying a set of peptides that can function as markers for peanut allergens. Emphasis was given to the identification of the three major peanut allergens Ara h 1, Ara h 2, and Ara h 3, because these proteins are considered to represent >30% of the total protein content of peanut and are directly relevant for the allergenic potential of this food. The analytical data obtained were used to perform databank searching in combination with de novo sequencing and led to the identification of a multitude of sequence tags for all three peanut allergens. Food processing such as roasting of peanuts is known to affect the stability of proteins and was shown to influence the detection of allergen sequence tags. The analysis of raw and roasted peanuts allowed the identification of five peanut-specific sequence tags that can function as markers of the specific allergenic proteins. For Ara h 1, two peptide markers were proposed, namely, VLEENAGGEQEER (m/z 786.88, charge 2+) and DLAFPGSGEQVEK (m/z 688.85, charge 2+), whereas for Ara h 2 only one peptide, RQQWELQGDR (m/z 439.23, charge 3+), was found to satisfy the required conditions. For Ara h 3, the two specific peptides, SPDIYNPQAGSLK (m/z 695.35, charge 2+) and SQSENFEYVAFK (m/z 724.84, charge 2+), were selected. Other peptides have been proposed as indicative for food processing.  相似文献   

19.
The objective of this study was to characterize the changes in peptide solubility resulting from changing some physicochemical conditions in a tryptic hydrolysate of beta-lactoglobulin (beta-LG). The turbidity (500 nm) of a 1% solution of tryptic peptides was measured at pH 3-10, at 5, 25, and 50 degrees C, in the presence of different salt concentrations (0, 0.5, and 1 M NaCl), in the presence of denaturing and reducing agents (6 M urea, 5% SDS, or 5% beta-mercaptoethanol), and under an electric field (isoelectric focusing). The results reveal an increase in turbidity of the peptide solution at pH 4, but a slight increase in turbidity was also observed at pH 8, which is attributable to peptides linked by disulfide bridges. The effect of temperature and ionic strength on the turbidity occurring at pH 4 indicates that mainly hydrophobic interactions are involved in the aggregation process. The material in the precipitate at pH 4 was identified as the peptides beta-LG 1-8, 15-20, and 41-60 and non-hydrolyzed alpha-lactalbumin. These results suggest that a limited number of peptides are involved in the aggregation process observed at pH 4, some of which having bioactive (beta-LG 15-20, ACE inhibitor, and opioid) or emulsifying properties (beta-LG 41-60). Aggregation of these peptides at acidic pH indicates that a simple acidification step could represent an easy process for isolating peptidic fractions enriched in bioactive or functional peptides.  相似文献   

20.
Synchrotron Fourier transform infrared (FTIR) microspectroscopy can explore molecular chemical features of the microstructure of feeds. The most straightforward method of data analysis is the mapping of specific functional group intensities and frequencies by peak heights and/or areas. However, this univariate statistical method does not always accurately identify functional group locations and concentrations, because the so-called "unique" bands for the peak area mapping have more or less inference with other nonunique bands in feed and plant tissues. The objective of this study was to use a multivariate analysis method--called agglomerative hierarchical cluster analysis (CLA)--to analyze infrared spectra for chemical imaging. The results show the CLA method gave satisfactory results and was conclusive in showing that it can discriminate and classify functional group differences existing in different structure regions. This approach (CLA for chemical imaging) places synchrotron FTIR microspectroscopy at the forefront of those techniques that could potentially be used in the rapid characterization of feed microstructure.  相似文献   

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