Functional region identification in proteins by accumulative-quantitative peptide mapping using RP-HPLC-MS

A new method was developed to identify regions in proteins from which peptides are derived with specific functional properties. This method is applicable for systems in which peptides of a hydrolyzed protein possess specific functional properties, but are too large to be sequenced directly and/or the peptide mixture is too complex to purify and characterize each peptide individually. In the present work, aggregating peptides obtained by proteolytic hydrolysis of soy glycinin were used as a case study. The aggregating peptides are isolated and subsequently further degraded with trypsin to result in peptides with a mass <5000 Da to enable sequence identification using RP-HPLC-MS in combination with MS/MS. Prior to RP-HPLC the peptides are fractionated using anion and cation exchange chromatography. The fractions obtained are analyzed with RP-HPLC-MS. The peptides, with identified sequences, were quantified using the peak areas of the RP-HPLC chromatograms measured at 214 nm. Next, the peak areas were corrected for the molar extinction coefficient of the individual peptides, followed by accumulative-quantitative peptide mapping. The results show that in complex systems, based on the method described, the regions in the parental protein from which the functional peptides originate can be properly identified.     

Heat denaturation of soy glycinin: influence of pH and ionic strength on molecular structure

The 7S/11S glycinin equilibrium as found in Lakemond et al. (J. Agric. Food Chem. 2000, 48, xxxx-xxxx) at ambient temperatures influences heat denaturation. It is found that the 7S form of glycinin denatures at a lower temperature than the 11S form, as demonstrated by a combination of calorimetric (DSC) and circular dichroism (CD) experiments. At pH 7.6, at which glycinin is mainly present in the 11S form, the disulfide bridge linking the acidic and the basic polypeptides is broken during heat denaturation. At pH 3.8, at which glycinin has dissociated partly into the 7S form, and at pH 5.2 this disruption does not take place, as demonstrated by solubility and gel electrophoretic experiments. A larger exposure of the acidic polypeptides (Lakemond et al., 2000) possibly correlates with a higher endothermic transition temperature and with the appearance of an exothermic transition as observed with DSC. Denaturation/aggregation (studied by DSC) and changes in secondary structure (studied by far-UV CD) take place simultaneously. Generally, changes in tertiary structure (studied by near-UV CD) occur at lower temperatures than changes in secondary structure.     

beta-Conglycinin and glycinin in high-protein soybean seeds

The agronomic performance and storage proteins of high seed protein lines of soybeans [Glycine max L. (Merr.)] were investigated to determine if the two major storage proteins, beta-conglycinin and glycinin, contribute to the increased protein content of high seed protein lines. Subunits of these two major storage proteins were estimated by scanning SDS-PAGE gels by scanning densitometry. The relative rankings of the lines with respect to seed size and protein content were not different between years in one environment over 5 years, but oil and total protein and oil contents and the ratio of protein to oil differed. The alpha', alpha, and beta subunits of beta-conglycinin were significantly higher in the high-protein lines except CX797-115, CX804-108, CX804-3, D81-8498, and NC-2-62. The acidic A(3) polypeptide of glycinin was significantly higher in high-protein lines except 76-48773, CX804-108, CX804-3, D81-8498, and NC-2-62, whereas the acidic polypeptides A(1,2,4) of glycinin were significantly higher in all of the high-protein lines. The basic polypeptides of glycinin were significantly higher in all high seed protein lines except D81-8259. In conclusion, high-protein lines appear to contain more beta-conglycinin and glycinin than normal-protein soybean lines, and the amounts of subunits and polypeptides differ among lines.     

Immunochemical studies on gastric and intestinal digestion of soybean glycinin and beta-conglycinin in vivo.

Two experiments were conducted to study gastric and small intestinal digestion of soybean glycinin and beta-conglycinin in preruminant calves fed milk replacers containing a mixture of skim milk powder and antigenic heated soybean flour. In experiment 1, duodenal passage of immunoreactive beta-conglycinin lasted for a much longer time after the morning meal than that of glycinin. Western blotting revealed the early abomasal outflow of glycinin subunits that associated nearly intact basic polypeptides to partially degraded acidic polypeptides. Intact beta-conglycinin was evidenced at most sampling times. In experiment 2, intact basic glycinin (M(r) = 21000) associated with partially digested acidic glycinin (7000 < M(r) < 25000) was demonstrated in ileal digesta up to 8-10 h after the meal. beta-Conglycinin immunoreactivity could not be evidenced by Western blotting in ileal digesta.     

Hydrolysis of whey protein isolate with Bacillus licheniformis protease: fractionation and identification of aggregating peptides

The objective of this work was to identify the dominant aggregating peptides from a whey protein hydrolysate (degree of hydrolysis of 6.8%) obtained with Bacillus licheniformis protease. The aggregating peptides were fractionated with preparative reversed-phase chromatography and identified with liquid chromatography-mass spectrometry. The results showed that the dominant aggregating peptide, at pH 7.0, was beta-lg AB [f1-45]. In addition, the peptides beta-lg AB [f90-108]-S-S-alpha-la [f50-113], alpha-la [f12-49]-S-S-alpha-la [f50-113], beta-lg AB [f90-108]-S-S-beta-lg AB [f90-108], beta-lg A [f90-157], and beta-lg AB [f135-157/158] were also identified as main aggregating peptides. The results further showed that aggregation, via hydrophobic interactions, prevented further digestion (at pH 8.0), thereby explaining the large size of the aggregating peptides. It is hypothesized that B. licheniformis protease breaks down hydrophilic segments in the substrate and, therefore, preserves hydrophobic segments that aggregate once exposed to the solvent.     

Prediction of molar extinction coefficients of proteins and peptides using UV absorption of the constituent amino acids at 214 nm to enable quantitative reverse phase high-performance liquid chromatography-mass spectrometry analysis

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.     

Soybean glycinin subunits: Characterization of physicochemical and adhesion properties

Soybean proteins have shown great potential for applications as renewable and environmentally friendly adhesives. The objective of this work was to study physicochemical and adhesion properties of soy glycinin subunits. Soybean glycinin was extracted from soybean flour and then fractionated into acidic and basic subunits with an estimated purity of 90 and 85%, respectively. Amino acid composition of glycinin subunits was determined. The high hydrophobic amino acid content is a major contributor to the solubility behavior and water resistance of the basic subunits. Acidic subunits and glycinin had similar solubility profiles, showing more than 80% solubility at pH 2.0-4.0 or 6.5-12.0, whereas basic subunits had considerably lower solubility with the minimum at pH 4.5-8.0. Thermal analysis using a differential scanning calorimeter suggested that basic subunits form new oligomeric structures with higher thermal stability than glycinin but no highly ordered structures present in isolated acidic subunits. The wet strength of basic subunits was 160% more than that of acidic subunits prepared at their respective isoelectric points (pI) and cured at 130 degrees C. Both pH and the curing temperature significantly affected adhesive performance. High-adhesion water resistance was usually observed for adhesives from protein prepared at their pI values and cured at elevated temperatures. Basic subunits are responsible for the water resistance of glycinin and are a good starting material for the development of water-resistant adhesives.     

Enzyme-induced gelation of extensively hydrolyzed whey proteins by Alcalase: peptide identification and determination of enzyme specificity

Extensive hydrolysis of whey protein isolate by Alcalase was shown to induce gelation mainly via hydrophobic interactions. The aim of this work was to characterize the peptides released in order to better understand this phenomenon. The apparent molecular mass distribution indicated that aggregates were formed by small molecular mass peptides (<2000 Da). One hundred and thirty peptides with various lengths were identified by reversed-phase high-performance liquid chromatography coupled with electrospray ionization mass spectrometry. Alcalase was observed to have a high specificity for aromatic (Phe, Trp, and Tyr), acidic (Glu), sulfur-containing (Met), aliphatic (Leu and Ala), hydroxyl (Ser), and basic (Lys) residues. Most peptides had an average hydrophobicity of 1-1.5 kcal/residue and a net charge of 0 at the pH at which gelation occurred (6.0). Therefore, an intermolecular attractive force such as hydrophobic interaction suggests the formation of aggregates that further leads to the formation of a gel.     

Surface functional properties of native, acid-treated, and reduced soy glycinin. 1. Foaming properties

Foaming properties of native and chemically modified glycinin were evaluated. Effects of ionic strength and glycinin composition and concentration on foam formation and stabilization were studied. Glycinin was modified by means of combined treatments: cold or hot acidic treatments, with or without later disulfide bridges reduction. Modified proteins obtained from glycinin present different degrees of dissociation, deamidation, and as consequence, varied surface hydrophobicity and molecular size. Parameters of forming and stabilizing of foam were correlated with both deamidation and dissociation degrees of modified and native glycinin samples. A positive relationship was observed between surface behavior and foaming properties of different protein species. Results show that dissociation, deamidation, and reduction have produced structural changes on glycinin (increased surface hydrophobicity, increased net charge, decreased molecular size) which enhance the adsorption and anchorage of proteins at the air-water interface and, consequently, improve the foam forming and stabilizing capacities.     

Surface functional properties of native, acid-treated, and reduced soy glycinin. 2. Emulsifying properties

Emulsifying properties of native and chemically modified soy glycinins were studied. The influence of ionic strength, protein sample composition and concentration, and assay conditions on the flocculation-creaming process and coalescence resistance was analyzed. Differences in these emulsifying properties were exhibited by native glycinins, which have a variable content of 4S, 11S, and 15S forms. Structure and functionality of native glycinin were modified by means of combined treatments: mild acidic treatments without heating or with heating at variable time and with or without disulfide bonds reduction. Modified glycinins presented different degrees of deamidation, surface hydrophobicity, and molecular mass. A slight enhancement of emulsifying stability at moderated deamidation degrees was observed. In different protein samples, a positive relationship between the flocculation-creaming rate constant and equilibrium oil volume fraction of emulsions with surface hydrophobicity was detected. A remarkable difference was observed between reduced and nonreduced samples, mainly with respect to behavior at low or high ionic strength.     

Hydrolysis of whey protein isolate with Bacillus licheniformis protease: aggregating capacities of peptide fractions

In a previous study, peptides aggregating at pH 7.0 derived from a whey protein hydrolysate made with Bacillus licheniformis protease were fractionated and identified. The objective of the present work was to investigate the solubility of the fractionated aggregating peptides, as a function of concentration, and their aggregating capacities toward added intact proteins. The amount of aggregated material and the composition of the aggregates obtained were measured by nitrogen concentration and size exclusion chromatography, respectively. The results showed that of the four fractions obtained from the aggregating peptides, two were insoluble, while the other two consisted of 1:1 mixture of low and high solubility peptides. Therefore, insoluble peptides coaggregated, assumedly via hydrophobic interactions, other relatively more soluble peptides. It was also shown that aggregating peptides could aggregate intact protein nonspecifically since the same peptides were involved in the aggregation of whey proteins, beta-casein, and bovine serum albumin. Both insoluble and partly insoluble peptides were required for the aggregation of intact protein. These results are of interest for the applications of protein hydrolysates, as mixtures of intact protein and peptides are often present in these applications.     

Characterization of methanol-soluble and methanol-insoluble proteins from developing peanut seed

The 85% methanol-soluble proteins are known to specifically contribute to the production of flavor of roasted peanut. To determine the nature of the 85% methanol-soluble proteins, they were isolated from the peanut seed, and the 85% methanol-soluble (MS) and 85% methanol-insoluble (MIS) fractions were characterized using polyacrylamide gel electrophoresis (PAGE) and capillary electrophoresis. The results showed that the 85% MS fraction contained lower amounts (9-10%) of protein than the MIS fraction (15-33%). Protein content of the MIS fraction increased more significantly during seed maturation than it did in the MS fraction. Unlike the protein, free amino acids and soluble sugars levels of the MS fraction decreased significantly during seed maturation. The 85% MS fraction contained predominantly low molecular weight (<20 kDa) proteins/polypeptides, whereas the MIS fraction contained a mixture of polypeptides with molecular weight between 14 kDa and 90 kDa. SDS-PAGE showed no major changes in the polypeptide composition of the MS fraction during seed maturation. Capillary electrophoretic analysis revealed major qualitative and quantitative changes in the protein and polypeptide composition of the MS and MIS fractions during seed maturation. Fatty acid analysis of these fractions indicated that the MS fraction is lipoprotein in nature and rich in oleic and linoleic acids.     

Effect of physicochemical conditions on peptide-peptide interactions in a tryptic hydrolysate of beta-lactoglobulin and identification of aggregating peptides

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.     

Positional effect on protein and oil content and composition of soybeans

Soybean (Glycine max [L.] Merr.) protein and oil qualities, with respect to monogastric nutrition, have been linked to the relative abundance of specific protein subunits and fatty acids, respectively. An analysis of field-grown soybean seeds by near-infrared spectroscopy revealed significant differences in their protein and oil contents as a function of nodal position. Seed proteins from the plant apex were high in protein and low in oil content, while those from the basal region exhibited an opposite pattern of accumulation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total seed proteins revealed that the beta-subunit of beta-conglycinin content was 4-fold higher in seeds from the apical nodes than in seeds from basal nodes. The glycinin A3 polypeptide content gradually increased in successively lower nodes from the top of the plant. Its accumulation was drastically reduced when nitrogen was applied at specific growth stages. Exogenous nitrogen did not alter the pattern of beta-subunit accumulation, but accrual of the acidic and basic polypeptides of glycinin was diminished. The remaining seed storage protein components were not influenced by nodal position or nitrogen application. Gas chromatographic analysis of fatty acids indicated that only oleic (18:0) and linoleic (18:2) acids showed variability in accumulation at different nodes. Neither the abundance nor the distribution of the fatty acids was altered by nitrogen application.     

Cloning and characterization of an 11S legumin, Car i 4, a major allergen in pecan

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.     

马面鱼皮胶原抗氧化肽的分离制备及稳定性研究

为促进对马面鱼皮资源的综合利用,开发高附加值产品,本试验以DPPH自由基清除率和水解度(DH)为评价指标,探讨马面鱼皮胶原蛋白的最佳酶解工艺,并采用超滤和凝胶柱层析法分离制备抗氧化肽,通过超高效液相色谱-质谱联用(UPLC-MS)法对其进行结构解析。此外,还探讨了pH值、温度及体外模拟消化对多肽抗氧化活性的影响。结果表明,利用双酶分步酶解法可制备高活性抗氧化多肽,即在底物浓度3%,加酶量3 600 U·g^-1以及温度50℃的条件下先用Proteasea A ‘Amano’2G酶解3 h,再用酸性蛋白酶酶解2 h,清除DPPH自由基的IC₅₀值为13.03 mg·mL ^-1。经超滤及柱层析分离后,可得到抗氧化活性较高的A₁组分,其清除DPPH自由基的 IC₅₀值为1.80 mg·mL^-1。稳定性研究结果表明,所制备的胶原蛋白抗氧化肽热稳定性好,在偏酸性条件下能保持较高的活性,经体外模拟胃肠消化后仍能保持较高的抗氧化活性。根据UPLC-MS分析推测A₁的氨基酸序列可能为Gly-Glu-Gly-Ala-Cys-Asn或Asn-Glu-Gly-Ala-Cys-Gly。本研究结果为马面鱼皮的高值化利用及高活性抗氧化肽的筛选提供了一定的理论依据。     

Characterization of storage proteins in wild (Glycine soja) and cultivated (Glycine max) soybean seeds using proteomic analysis

A combined proteomic approach was applied for the separation, identification, and comparison of two major storage proteins, beta-conglycinin and glycinin, in wild (Glycine soja) and cultivated (Glycine max) soybean seeds. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with three different immobilized pH gradient (IPG) strips was an effective method to separate a large number of abundant and less-abundant storage proteins. Most of the subunits of beta-conglycinin were well-separated in the pH range 3.0-10.0, while acidic and basic glycinin polypeptides were well-separated in pH ranges 4.0-7.0 and 6.0-11.0, respectively. Although the overall distribution pattern of the protein spots was similar in both genotypes using pH 3.0-10.0, variations in number and intensity of protein spots were better resolved using a combination of pH 4.0-7.0 and pH 6.0-11.0. The total number of storage protein spots detected in wild and cultivated genotypes was approximately 44 and 34, respectively. This is the first study reporting the comparison of protein profiles of wild and cultivated genotypes of soybean seeds using proteomic tools.     

Identification of peptides in aggregates formed during hydrolysis of beta-lactoglobulin B with a Glu and Asp specific microbial protease

The purpose of the present study was to identify the peptides responsible for aggregate formation during hydrolysis of beta-lactoglobulin by BLP at neutral pH. Hydrolysates taken at various stages of aggregate formation were separated into a precipitate and a soluble phase and each was analyzed by CE and mass spectrometry. The aggregates consisted of six to seven major peptides of which four were tentatively identified. The peptides were positively charged at neutral pH and had a high charge-to-mass ratio at low pH. The fragment f135-158 seemed to be the initiator of aggregation, since it was present at high concentration in the aggregates at all stages, and the concentration of this peptide remained low in the supernatant. F135-158 contains several basic and acid amino acids alternating with hydrophobic amino acids, which is in accordance with formation of noncovalently linked aggregates, as previously shown.     

Hydrophobicity of bitter peptides from soy protein hydrolysates

Soy peptides were characterized for flavor, chemical properties, and hydrophobicity to investigate their relationships with bitterness. Five peptide fractions ranging in average molecular mass from 580 to 11300 Da were fractionated by ultrafiltration from two commercial soy protein hydrolysates. The bitterness of fractionated peptides was related to molecular mass, with maximum bitterness observed at approximately 4000 Da for one hydrolysate and 2000 Da for the other. The bitterness increased as the peptide M(w) decreased to 3000 Da for the first hydrolysate and to 2000 Da for the second one and then decreased as the peptide M(w) decreased below 1000 Da. The peptide fraction with molecular mass of <1000 Da showed the lowest bitterness for both. The hydrophobicity data based on Q values do not support Ney's Q rule as a predictor of bitterness for soy peptides.     

Antioxidant activity of peptides obtained from porcine myofibrillar proteins by protease treatment

Hydrolysates obtained from porcine myofibrillar proteins by protease treatment (papain or actinase E) exhibited high antioxidant activity in a linolenic acid peroxidation system induced by Fe(2+). Hydrolysates produced by both papain and actinase E showed higher activities at pH 7.1 than at pH 5.4. The antioxidant activity of the papain hydrolysate was almost the same as that of vitamin E at pH 7.0. These hydrolysates possessed 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity and chelating activity toward metal ions. Antioxidant peptides were separated from the papain hydrolysate by ion exchange chromatography. The acidic fraction obtained by this method exhibited higher activity than the neutral or basic fractions. Antioxidant peptides in the acidic fraction were isolated by high-performance liquid chromatography on an ODS column and shown to possess the structures DSGVT, IEAEGE, DAQEKLE, EELDNALN, and VPSIDDQEELM. The DAQEKLE peptide showed the highest activity among these peptides.