Identification of proteins of the yeast protein map using genetically manipulated strains and peptide-mass fingerprinting

In this study we used genetically manipulated strains in order to identify polypeptide spots of the protein map of Saccharomyces cerevisiae. Thirty-two novel polypeptide spots were identified using this strategy. They corresponded to the product of 23 different genes. We also explored the possibilities of using peptide-mass fingerprinting for the identification of proteins separated on our gels. According to this strategy, proteins contained in spots are digested with trypsin and the masses of generated peptides are determined by matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). The peptide masses are then used to search a yeast protein database for proteins that match the experimental data. Application of this strategy to previously identified polypeptide spots gave evidence of the feasibility of this approach. We also report predictions on the identities of nine unknown spots using MALDI-MS.     

Quantitative comparative phosphoproteomic analysis of the effects of colostrum and milk feeding on liver tissue of neonatal calves

Posttranslational modifications, mostly phosphorylation, are critical for protein structure and function. However, the association between liver phosphoproteins in neonatal calves and colostrum intake is not well understood. In this study, we examined the liver phosphoproteome profile in neonatal calves after receiving colostrum or milk. Liver tissue samples were collected from control calves (CON, n = 3) 2 h after birth and from calves that received colostrum (CG, n = 3) or milk (MG, n = 3) 24 h after birth. Hepatic phosphoprotein expression profiles were analyzed using quantitative proteomics based on the liquid chromatography–tandem mass spectrometry method. In total, 1,587 phosphorylated sites were identified in 1,011 liver proteins. The most abundant phosphorylation site AA was serine (87.5%), followed by threonine (11.9%) and tyrosine (0.5%). Among the 1,011 phosphoproteins, 219, 453, and 26 displayed differential expression in the CG versus MG, CG versus CON, and MG versus CON comparisons, respectively. Differentially expressed phosphoproteins in the CG–MG comparison included 3-phosphoinositide-dependent protein kinase 1, glucose transporter member 4, protein kinase N2, and vinculin, which were mainly involved in the glycogen metabolic process, transport, growth and development, and cell adhesion process, according to Gene Ontology analysis. Pathway analysis indicated their enrichment in the insulin signaling pathway, spliceosome, and adherens junction. The CG–CON comparison identified differentially expressed phosphoproteins and their target genes that were largely involved in the cellular process, macromolecule metabolic process, developmental process, and transport. Pathway analysis indicated their association with endocytosis, mechanistic target of rapamycin, AMP-activated protein kinase, and insulin signaling pathways. These data demonstrate that changes in the phosphoproteins of liver tissues may play an important role in energy metabolism and immune response in the calves that received colostrum. These results provide novel insights into the crucial roles of protein phosphorylation during the early life of newborn calves.     

Proteomic analysis of processing by-products from canned and fresh tuna: Identification of potentially functional food proteins

Proteomic approaches have been used to identify the main proteins present in processing by-products generated by the canning tuna-industry, as well as in by-products derived from filleting of skeletal red muscle of fresh tuna. Following fractionation by using an ammonium sulphate precipitation method, three proteins (tropomyosin, haemoglobin and the stress-shock protein ubiquitin) were identified in the highly heterogeneous and heat-treated material discarded by the canning-industry. Additionally, this fractionation method was successful to obtain tropomyosin of high purity from the heterogeneous starting material. By-products from skeletal red muscle of fresh tuna were efficiently fractionated to sarcoplasmic and myofibrillar fractions, prior to the identification based mainly on the combined searching of the peptide mass fingerprint (MALDI–TOF) and peptide fragment fingerprinting (MALDI LIFT-TOF/TOF) spectra of fifteen bands separated by 1D SDS–PAGE. Thus, the sarcoplasmic fraction contained myoglobin and several enzymes that are essential for efficient energy production, whereas the myofibrillar fraction had important contractile proteins, such as actin, tropomyosin, myosin or an isoform of the enzyme creatine kinase. Application of proteomic technologies has revealed new knowledge on the composition of important by-products from tuna species, enabling a better evaluation of their potential applications.     

Major proteins of the goat milk fat globule membrane

Fat is present in milk as droplets of triglycerides surrounded by a complex membrane derived from the mammary epithelial cell called milk fat globule membrane (MFGM). Although numerous studies have been published on human or bovine MFGM proteins, to date few studies exist on MFGM proteins from goat milk. The objective of this study was thus to investigate the protein composition of the goat MFGM. Milk fat globule membrane proteins from goat milk were separated by 6% and 10% sodium dodecyl sulfate-PAGE and were Coomassie or periodic acid-Schiff stained. Most of MFGM proteins [mucin-1, fatty acid synthase, xanthine oxidase, butyrophilin, lactadherin (MFG EGF-8, MFG-E8), and adipophilin] already described in cow milk were identified in goat milk using peptide mass fingerprinting. In addition, lectin staining provided a preliminary characterization of carbohydrate structures occurring on MFGM proteins from goat milk depending on α_S1-casein genotype and lactation stage. We provide here first evidence of the presence of O-glycans on fatty acid synthase and xanthine oxidase from goat milk. A prominent difference between the cow and the goat species was demonstrated for lactadherin. Indeed, whereas 2 polypeptide chains were easily identified by peptide mass fingerprinting matrix-assisted laser desorption/ionization-time of flight analysis within bovine MFGM proteins, lactadherin from goat milk consisted of a single polypeptide chain. Another striking observation was the presence of caseins associated with MFGM preparations from goat milk, whereas virtually no caseins were found in MFGM extracts from bovine milk. Taken together, these observations strongly support the existence of a singular secretion mode previously hypothesized in the goat.     

翡翠贻贝副肌球蛋白的特性及在模拟胃肠液中的消化

为探究贝类副肌球蛋白（paramyosin,PM）的热稳定性、pH值稳定性及其在模拟胃肠液中的消化特性,以翡翠贻贝（Perna viridis）为对象,利用硫酸铵盐析和羟基磷灰石柱层析等方法,从肌肉中纯化PM,采用肽质量指纹图谱法对其进行鉴定,利用圆二色谱测定其二级结构及热变性温度。结果显示,翡翠贻贝PM分子质量为99.5 kDa;肽质量指纹图谱分析获得26 个肽段,共330 个氨基酸残基,与地中海贻贝(Mytilus galloprovincialis)PM的序列一致性达到100%,表明纯化的蛋白为PM。圆二色谱结果显示,PM呈现典型的α-螺旋结构,其热变性温度为（56.3±0.2）℃。在30～100 ℃热处理30 min,PM未出现沉淀聚集现象,在pH 6～11范围内也较稳定,但当pH≤5时稳定性差,出现沉淀聚集现象。与胃蛋白酶、胰蛋白酶及胰凝乳蛋白酶对PM的单独消化作用相比,3 种酶连续作用可使PM有效降解,但仍有分子质量30 kDa的片段未被完全消化。本研究表明,翡翠贻贝PM具有较好的耐热性及耐消化性,为贻贝深加工及PM的后续研究提供一定理论参考。     

Characterization of major milk proteins from BALB/c and C3H mice.

Milk proteins from BALB/c and C3H mice were characterized with respect to their electrophoretic migration in polyacrylamide gels under alkaline and acid conditions. The major casein and whey proteins from each strain migrated similarly under the conditions employed. Phosphoproteins were identified by staining with "Stains-all" and by changes in electrophoretic mobility and staining induced by prior treatment with phosphatase. Sialic acid-rich glycoproteins were identified by staining with periodic acid-Schiff and with "Stains-all" by prior treatment with neuraminidase to identify sialic acid as the acidic portion of the molecule. The two major whey proteins were characterized further by their migration in sodium dodecyl sulfate gels. One protein had the same mobility as mouse serum albumin. The other protein migrated with a mobility similar to that of bovine alpha-lactalbumin. The identity of the former protein was confirmed by its reaction with goat anti-mouse serum albumin in an immunodiffusion procedure, and the latter protein by its B protein activity in the lactose synthetase assay.     

蓝圆鲹骨骼肌脯氨酸内肽酶的分离纯化及其对胶原肽的作用

以蓝圆鲹为研究对象,探讨脯氨酸内肽酶(Prolyl endopeptidase,PEP)对鱼类肌肉胶原蛋白的作用及其机理。通过硫酸铵分级盐析,DEAE-Sephacel阴离子交换,Phenyl-Sepharose疏水层析和Q-Sepharose阴离子交换,从蓝圆鲹骨骼肌中分离纯化得到一种脯氨酸内肽酶。SDS-PAGE结果显示,PEP的分子量为82 ku,肽质量指纹图谱分析得到16个肽片段,共169个氨基酸残基。片段序列与墨西哥鲷鱼(Neolamprologus brichardi)PEP的同源性达98.8%,证明纯化得到的酶是PEP。PEP的最适温度为35℃,但热稳定性较差;最适p H为6.0,在p H 5.0~7.5之间有较好的稳定性。将PEP与合成的鱼胶原蛋白小肽反应,利用反相高效液相色谱对产物进行分离,电喷雾质谱分析结果显示PEP的水解位点在脯氨酸残基的羧基端。以上结果表明,鱼类肌肉中的PEP能够协同金属蛋白酶,通过切割脯氨酸残基进一步降解胶原小肽从而参与到鱼肌肉胶原蛋白的新陈代谢中,是鱼死后参与胶原蛋白降解的重要酶类。     

Bos d 13, A Novel Heat-Stable Beef Allergen

Scope

Red meat, a staple food of Western diets, can also induce IgE-mediated allergic reactions. Yet, apart from the heat-labile protein serum albumin and the carbohydrate α-Gal, the molecules causing allergic reactions to red meat remain unknown.

Methods and results

IgE reactivity profiles of beef-sensitized individuals are analyzed by IgE-immunoblotting with protein extracts from raw and cooked beef. Two IgE-reactive proteins are identified by peptide mass fingerprinting as myosinlight chain 1 (MYL1) and myosin light chain 3 (MYL3) in cooked beef extract and are designated Bos d 13 isoallergens. MYL1 and MYL3 are produced recombinantly in Escherichia coli. ELISAs proved their IgE reactivity and circular dichroism analysis showed that they represent folded molecules with remarkable thermal stability. In vitro gastrointestinal digestion experiments showed the higher stability of rMYL1 as compared to rMYL3. Exposure of a monolayer of Caco–2 cells to rMYL1 indicated that the molecule is able to cross intestinal epithelial cells without disturbing the integrity of the tight junctions, suggesting the sensitizing capacity of MYL1.

Conclusion

MYLs are identified as novel heat-stable bovine meat allergens.     

Proteome profiling-pitfalls and progress

In this review we examine the current state of analytical methods in proteomics. The conventional methodology using two-dimensional electrophoresis gels and mass spectrometry is discussed, with particular reference to the advantages and shortcomings thereof. Two recently published methods which offer an alternative approach are presented and discussed, with emphasis on how they can provide information not available via two-dimensional gel electrophoresis. These two methods are the isotope-coded affinity tags approach of Gygi et al. and the two-dimensional liquid chromatography-tandem mass spectrometry approach as presented by Link et al. We conclude that both of these new techniques represent significant advances in analytical methodology for proteome analysis. Furthermore, we believe that in the future biological research will continue to be enhanced by the continuation of such developments in proteomic analytical technology.     

鸡蛋清磷酸化蛋白质组鉴定与分析

为了系统地阐明鸡蛋清磷酸化蛋白质的结构和功能特性,本研究采用蛋白质组学策略对鸡蛋清的磷酸化修饰蛋白质组进行鉴定与分析。鸡蛋清酶解产物首先经固定化金属螯合亲和层析分离富集磷酸肽,再经纳升液相色谱-串联质谱分析和鉴定。通过数据库检索匹配,共鉴定出33 个特异性磷酸化修饰多肽,包含41 个磷酸化修饰位点,归属于25 种鸡蛋清磷酸化蛋白。基序分析显示,大多数磷酸化修饰位点的序列特征为“S-X-E”;基因本体功能注释分析表明,鸡蛋清磷酸化蛋白质主要参与“生物调节”、“刺激反应”、“发育过程”等生物学过程,主要涉及“结合”、“催化”等分子功能。本研究结果将为鸡蛋清蛋白质相关研究提供关键的磷酸化修饰结构信息。     

Biochemical and gelling properties of tilapia surimi and protein recovered using an acid-alkaline process

The biochemical and gel properties of tilapia surimi prepared by a conventional washing method and protein isolated using alkaline-acid-aided processes were studied. Solubility and recovery of protein was found to be highest by using a conventional method, followed by an alkaline- and acid-aided process, respectively. Decreases in myoglobin and lipid contents were found in alkaline- or acid-aided process when compared to the conventional process (p < 0.05). The highest breaking force and deformation of kamaboko and modori gels was found in the gels prepared by the conventional washing method. Higher expressible water and whiteness were found in modori gels when compared to kamaboko gels. TCA-soluble peptide contents of conventional surimi gels were lower than those of acid- and alkaline-recovered protein gels. Degradation of myofibrillar protein was observed in acid-isolated protein. Microstructure of kamaboko gels showed more compact network than in modori gels in both conventional surimi and protein recovered using the pH-shift process.     

鹰嘴豆抗氧化肽的分离纯化、鉴定及其抗氧化活性

以发芽鹰嘴豆为原料,制备鹰嘴豆抗氧化肽,采用葡聚糖凝胶对其进行分离纯化,通过测定自由基清除能力、还原力、脂质氧化抑制能力、对自由基诱导的蛋白质和DNA损伤的保护能力评价纯化肽的抗氧化活性。对纯化肽进行鉴定合成以验证其抗氧化活性,并对合成多肽进行安全性预测。结果表明,纯化后的多肽有较好的自由基清除能力、还原力,且对亚油酸自氧化有明显的抑制作用,对自由基诱导的蛋白质和DNA损伤有保护作用。经预测,合成多肽无毒性与致敏性。综上,发芽鹰嘴豆水解物具有抗氧化能力,可用来制备抗氧化肽,从而增加鹰嘴豆的利用价值。     

与浓缩苹果汁后混浊有关的蛋白质组成分析

从新鲜苹果和浓缩苹果汁中分离纯化蛋白质，用肽质量指纹谱对其进行鉴定，测定氨基酸组成和多酚及多糖的含量，结果表明，新鲜苹果蛋白的丰要组分为Thaumatin-1ike protein1，是一糖蛋白，其疏水性氨基酸含量高，易引起苹果汁的后混浊。浓缩苹果汁中蛋白组分比较复杂，在贮存过程中与多酚及多糖发生结合，使分子量增大，其分子量均大于新鲜苹果蛋白。     

辣木籽抗氧化肽的分离鉴定及其稳定性分析

采用碱性蛋白酶酶解辣木籽蛋白制备抗氧化肽,通过超滤、Sephadex G-15葡聚糖凝胶柱层析分离纯化辣木籽抗氧化肽,利用LC-MS/MS鉴定其抗氧化肽序列,并分析pH、金属离子、温度对辣木籽抗氧化肽活性的影响.结果表明:当酶解物、超滤物(<3 kDa)、柱层析纯化物的浓度为8 mg/mL时,对应的还原力吸光值分别为0...     

High Pressure Effects on Gelation of Surimi and Turkey Breast Muscle Enhanced by Microbial Transglutaminase

High pressure effects on the strength (stress) and elasticity/deformability (strain) of surimi and turkey breast meat gels containing microbial transglutaminase (TGase) were evaluated. Pressurization of muscle proteins at 4°C prior to incubation at 25°C or 40°C (setting) increased gel strength 2–3 fold in uncooked surimi gels, but not in uncooked turkey gels. However, pressurization at 40°C or 50°C prior to setting increased the strength of turkey gels. Similar effects of prior pressurization, but of lesser magnitude, occurred in gels formed by directly or subsequently (following setting) cooking at 90°C. SDS-PAGE confirmed that myosin crosslinking occurred due to TGase activity during the setting treatment, which had survived prior pressure treatment. High pressure rendered protein substrates more accessible to TGase thereby enhancing intermolecular cross-link formation and gel strength.     

Evaluation of different methods of protein extraction and identification of differentially expressed proteins upon ethylene-induced early-ripening in banana peels

BACKGROUND: Banana peels (Musa spp.) are a good example of a plant tissue where protein extraction is challenging due to the abundance of interfering metabolites. Sample preparation is a critical step in proteomic research and is critical for good results. RESULTS: We sought to evaluate three methods of protein extraction: trichloroacetic acid (TCA)‐acetone precipitation, phenol extraction, and TCA precipitation. We found that a modified phenol extraction protocol was the most optimal method. SDS‐PAGE and two‐dimensional gel electrophoresis (2‐DE) demonstrated good protein separation and distinct spots of high quality protein. Approximately 300 and 550 protein spots were detected on 2‐DE gels at pH values of 3–10 and 4–7, respectively. Several spots were excised from the 2‐DE gels and identified by mass spectrometry. CONCLUSIONS: The protein spots identified were found to be involved in glycolysis, the tricarboxylic acid cycle, and the biosynthesis of ethylene. Several of the identified proteins may play important roles in banana ripening. Copyright © 2012 Society of Chemical Industry     

Isolation and Heat Stability of Trypsin Inhibitors in Amaranth (Amaranthus hypochondriacus)

Trypsin inhibitors were isolated from seeds of Amaranthus hypochondriacus by extraction at pH 7.5, heat treatment at 70°C for 15 min and affinity chromatography with trypsin-Sepharose 4B. Inhibitors of trypsin and chymotrypsin were identified in polyacrylamide gels following electrophoresis using a stain procedure that employed the chromogenic substrate acetyl-D, L-phenylalanine-2-napthyl ester. Three of the thirteen trypsin inhibitors identified in the electrophoresis gels, also had antichymotryptic activity. The inhibitor preparation was very thermostable retaining 20% of its original activity after 7 hr at 100°C.     

Bitterness intensity of soybean protein hydrolysates—chemical and organoleptic characterization

Soluble and isoelectric soluble soybean protein hydrolysates were prepared by Alcalase treatment to a degree of hydrolysis of 3–15%. The bitterness intensity of the hydrolysates obtained was assessed on a five-point scale. The average relative molecular masses of peptides in the soluble hydrolysates (2250-1400) and isoelectric soluble hydrolysates (1313-800) and their hydrophobic peptide fractions (575-400) were determined by the trinitrobenzenesulphonic acid method. The molecular mass distribution of peptides in soluble and isoelectric soluble soybean hydrolysates and their hydrophobic peptide fractions was determined by gel permeation HPLC using a Zorbax Bio Series GF-250 column. The results suggest that the main reason for the bitterness of soybean protein hydrolysates prepared by Alcalase treatment are hydrophobic bitter peptides of relative molecular mass less than 1000.     

直接进样-质谱检测指纹图谱技术结合化学计量学研究干燥方法对黄花菜质量的影响

采用直接进样-质谱检测指纹图谱技术(flow injection mass spectrometric fingerprinting method,FIMS)结合化学计量学方法研究不同干燥方法处理黄花菜质量的影响。自然干燥、热风干燥、真空冷冻干燥3种处理后的30个黄花菜样品经简单提取后,不经色谱柱分离,直接进入质谱仪进行分析,FIMS分析1个样品仅需2 min,采集m/z 100~1000的离子响应强度信息,经预处理后由分析软件Simca-P进行主成分分析(PCA)和偏最小二乘法判别分析(PLS-DA)。结果表明负离子模式下m/z 191.06、259.09、341.11、503.16、421.15、609.15、665.21、711.22、827.27及989.32等离子出现高的响应强度。经PCA和PLS-DA分析得到的分布图将不同干燥处理的黄花菜聚为3类,反应了其化学成分的差异性;采用PCA处理后3个主成分的贡献率是91.2%,采用PLS-DA的分类模型预测能力达到97.3%。通过载荷图则可发现在分类过程中起决定作用的化学标记物。该方法作为一种极具特点的指纹图谱分析技术,可以用于不同加工方式的农产品质量快速判别研究。     

Separation and identification of zinc-chelating peptides from sesame protein hydrolysate using IMAC-Zn and LC–MS/MS

The metal chelating peptides from sesame protein hydrolysates (SPH), treated by papain, alcalase and trypsin, respectively, were investigated. The hydrolysates treated by trypsin had the highest metal chelating ability. The metal chelating peptides were isolated from the trypsin hydrolysates using immobilized metal affinity chromatography (IMAC-Zn²⁺). Further, six zinc-chelating peptides were identified with reversed phase (RP)-HPLC and mass spectrometry (LC–MS/MS). Three of these metal-chelating peptides, Ser-Met, Leu-Ala-Asn and Asn-Cys-Ser, were synthesized and the metal-chelating ability of peptides was measured. The Asn-Cys-Ser peptide showed the highest zinc and iron chelating ability, which was even higher than reduced glutathione (GSH). The results confirm that the zinc or iron chelating activity of these peptides, and provide further support to its feasibility as natural metal chelating agents from sesame protein.