基于iTRAQ技术分析牛初乳与常乳乳清差异蛋白质组

为阐明牛初乳、牛常乳乳清蛋白的差异,利用同位素标记相对和绝对定量(isobaric tags for relative and absolute quantitation,iTRAQ)蛋白质组学技术对二者进行蛋白质组差异分析,在得到的599 种具有定量信息的乳清蛋白中,鉴定出60 种差异蛋白.将牛初乳与牛常乳乳清丰度差...     

牛初乳与牛乳中乳清蛋白质组成及功能的对比分析

乳清富含多种功能特性和生物活性的蛋白质,本研究利用SDS-PAGE电泳将牛初乳与牛乳中乳清蛋白质的组成部分进行分离鉴定,发现牛初乳与牛乳中乳清蛋白质的组成存在较大的差异,且在牛初乳乳清中鉴定出290种蛋白,牛乳乳清中鉴定出325种蛋白。由GO功能注释分析发现,在生物过程中,牛初乳乳清蛋白在细胞定位建立和细胞定位中的作用略高于牛乳乳清蛋白。在分子功能上酶抑制活性作用是牛初乳乳清蛋白和牛乳中乳清蛋白的主要分子功能。在细胞组成上牛初乳乳清蛋白参与较多的是细胞外部分和细胞外空隙,与牛乳乳清蛋白相比参与的细胞组成大体相同。通过KEGG代谢通路分析可知,牛初乳和牛乳乳清蛋白均参与过补体及凝血级联反应通路。对牛初乳乳清蛋白组成进行研究,不仅能够增加牛初乳的利用率,并且为日后以乳清蛋白作为原料生产乳制品提供理论依据。     

不同泌乳期羊乳和牛乳的高通量定量乳清蛋白质组学

利用高分辨质谱技术获取牛初乳、牛常乳、羊初乳和羊常乳的乳清蛋白组轮廓,基于其非标记定量强度建立偏最小二乘判别分析模型。结果发现牛初乳和牛常乳在蛋白质组成方面较羊初乳和羊常乳更相似,同时筛选出羊乳中丰度较高的9 种蛋白用作标志物区分这4 组乳样。生物信息学分析发现羊乳中的高丰度蛋白大部分与免疫应答和代谢过程有关,说明羊乳更有助于新生儿建立抗微生物感染的免疫系统。该研究可加深对羊乳蛋白的认识,对牛乳及其制品的营养改良和母乳替代品的生产具有重要意义。     

牛乳中不同部分蛋白质的组成及功能分析

本研究通过SDS-PAGE电泳将牛乳中不同蛋白质组成部分进行分离鉴定发现,乳脂肪球膜中存在201种蛋白,乳清中存在96种蛋白,酪蛋白中存在21种蛋白,乳粒中存在43种蛋白,其中有27种相同表达的蛋白。通过GO功能注释分析发现,在生物过程中乳脂肪球膜蛋白发挥的作用大于乳清、乳粒蛋白,尤其是生物的调控作用;在分子功能上,牛乳蛋白的主要分子功能是结合作用,其中乳脂肪球膜蛋白的结合作用最强;而乳粒蛋白参与的转运活性分子功能大于乳脂肪球膜、乳清蛋白。在细胞组成上,与乳清、乳粒蛋白相比乳脂肪球膜蛋白参与的细胞组成均较多,而在细胞膜的组成上乳粒蛋白参与较多。通过京都基因与基因组百科全书(KEGG)代谢通路分析可知,乳脂肪球膜、乳清、乳粒中的蛋白均参与过氧化物酶体增殖物激活受体信号通路。对牛乳蛋白质组成进行研究,不仅能够增加牛乳的利用率,并且为日后以乳脂肪球膜、乳粒蛋白作为原料生产乳制品提供理论依据。     

基于生物质谱技术解析食源性糖蛋白糖基化位点的研究进展

糖蛋白作为生物体内重要的生物大分子,参与细胞的识别、粘着及迁移,并调控细胞的增殖及分化。目前已有大量研究表明,糖蛋白广泛存在于食品原料中,且发挥多种生理活性。鉴于糖蛋白糖基化的复杂性,解析糖基化位点成为糖蛋白结构表征的关键。本文对糖蛋白N-糖基化、O-糖基化以及部分糖链结构进行介绍,以N-糖基化作为主要研究对象,对核磁共振技术和质谱技术这两种糖基化位点的检测方法进行对比分析,归纳获得去糖基化和完整糖肽检测两种糖基化位点的检测方法,并阐释质谱碎裂方式、去糖基化分析机制以及基于糖肽的糖基化位点解析机理。本文旨在为通过生物质谱技术确定糖基化位点的策略提供参考,并为深入研究食源性糖蛋白的性质和功能奠定基础。     

不同泌乳期人乳与牛乳中游离氨基酸的对比

利用同位素标记相对和绝对定量技术结合高效液相色谱-串联质谱技术对不同泌乳期人乳与牛乳中游离氨基酸的种类及含量进行检测及对比分析。结果表明,人乳中游离氨基酸种类较牛乳更为丰富,含量也高于牛乳,且随着泌乳时间的延长其总量呈下降趋势。牛初乳、牛常乳、人初乳和人常乳中游离氨基酸总量分别为0.32、0.16、0.63?g/L和0.37?g/L。实验测定的42?种游离氨基酸中,人常乳中检出35?种,牛常乳中测得31?种,其中人常乳中有25?种游离氨基酸的含量高于牛常乳,人乳中组氨酸、苯丙氨酸、苏氨酸等含量显著高于牛乳（P＜0.05）,游离谷氨酸在人初乳、人常乳、牛常乳中含量均为最高,而牛初乳中游离牛磺酸含量最高。本研究分析了人乳、牛乳中游离氨基酸种类和含量的差异,可为详细的研究母乳氨基酸功能和氨基酸代谢组学提供了一定的理论依据,也可为生产婴幼儿奶粉和功能性乳制品提供参考。     

中国北方人初乳、牛初乳、牛常乳、牛血中胰岛素样生长因子-1和神经生长因子含量的比较

目的检测人初乳、集中饲养的新西兰进口荷斯坦乳牛的初乳、常乳和血液中胰岛素样生长因子-1(IGF-1)和神经生长因子(NGF)的含量,比较人初乳和牛初乳、牛初乳和牛常乳以及牛乳和同期采集的血液中IGF-1、NGF的含量。方法采用放射免疫试剂盒测定人初乳、牛初乳、牛常乳和血中IGF-1、NGF的含量,使用SPSS 13.0进行统计分析。结果本研究中测定的人初乳中IGF-1的含量是26.91μg/L,荷斯坦乳牛的初乳、常乳和血清中IGF-1的含量分别是38.40、20.14和37.35μg/L。人初乳中NGF的含量是300.47 ng/L,荷斯坦乳牛的初乳、常乳和血清中NGF的含量分别是69.82、110.37和9.63 ng/L。经统计学分析发现牛初乳和人初乳中IGF-1的含量差异没有显著性,牛初乳中IGF-1的含量高于牛常乳(P<0.05),牛乳中IGF-1的含量与同期血液中IGF-1的含量差异没有显著性。人初乳中NGF的含量高于牛初乳(P<0.05),牛初乳和牛常乳中NGF的含量差异没有显著性,同期牛血中NGF的含量低于牛乳中NGF的含量(P<0.05)。结论本研究发现牛初乳与人初乳IGF-1含量无明显差别,...     

牛初乳与牛常乳乳脂肪球膜中丰度差异蛋白的表征与分析

为阐明不同泌乳期间牛乳乳脂肪球膜蛋白的差异,选定牛初乳和牛常乳中的乳脂肪球膜蛋白作为研究对象,利用定量蛋白质组学技术对其中的蛋白质进行表征,并对两组间的丰度差异蛋白进行筛选及生物信息学分析。本研究共鉴定到763 种蛋白,其中197 种为两组共有蛋白,进一步从其中筛选出80 种丰度差异蛋白,包括41 种上调蛋白和39 种下调蛋白（牛初乳/牛常乳）。对上述丰度差异蛋白进行生物信息学分析发现,这些蛋白参与的主要细胞组分为细胞外泌体、细胞外空间、细胞外区域等,参与的主要代谢通路为代谢途径、嘌呤代谢、苯丙氨酸代谢、酪氨酸代谢、丙酮酸代谢、糖酵解/糖异生等,并进一步筛选出31 种与其他蛋白存在相互作用的关键蛋白,包括结合珠蛋白、2-磷酸-D-甘油酸水解酶等。研究结果将有助于了解泌乳期间乳蛋白成分的差异及其功能性,为牛乳制品的精深加工奠定基础。     

羊乳乳清蛋白组成和功能及其与人乳、牛乳的对比分析

本研究将羊乳、牛乳、人乳中乳清蛋白进行分离并结合液质联用技术鉴定,在羊乳、牛乳、人乳乳清蛋白中分别鉴定出156、278、454种蛋白质。与牛乳与人乳乳清蛋白对比显示,羊乳含有99种特异性表达蛋白质,与牛乳和人乳分别有31种和15种相同表达蛋白质。通过分析基因本体(gene ontology,GO)功能注释发现,羊乳乳清蛋白在生物过程中主要发挥生物调节作用;在分子功能上,主要体现在结合作用方面;在细胞组成上,参与的细胞组成主要为细胞器区和胞外区。羊乳乳清蛋白在以上三种功能上与人乳有较大差距,但与牛乳相近。通过分析京都基因与基因组百科全书系统(Kyoto Encyclopedia of Genes and Genomes,KEGG)代谢通路可知,羊乳主要参与补体和凝血级联反应以及吞噬作用,对人体免疫能力有积极影响。对羊乳与人乳、牛乳乳清蛋白组成及功能区别的研究,为羊乳的进一步研究和开发提供一定的理论参考。     

驴乳中蛋白质组分的分离纯化与鉴定

目的：分离纯化并鉴定驴乳乳清中蛋白质组分,为进一步研究驴乳用于辅助治疗疾病,以及为作为人乳替代品提供参考。方法：采用DEAE-52 离子交换层析和Sephadex G-100 凝胶层析分离纯化驴乳乳清中蛋白质组分,并通过十二烷基硫酸钠--聚丙烯酰胺凝胶电泳法和高效凝胶渗透色谱法对所纯化蛋白质进行鉴定。结果：与牛乳乳清中蛋白组分相对比,发现驴乳乳清中存在3 种未知蛋白质,分子质量分别为32、70.1、72.2kD。结论：驴乳中除了含有与牛乳相似的营养成分外,还具有其他生物活性成分,它们可能是一些保护性蛋白,在机体的抗病机制方面起着重要作用。     

驴初乳与常乳乳脂球膜蛋白质组的对比分析

为阐明驴初乳与常乳中乳脂球膜蛋白的差异,利用蛋白质组学技术对二者进行蛋白质组差异分析。在驴初乳和驴常乳中分别鉴定到216 种和215 种乳脂球膜蛋白,并在其中筛选出40 种差异蛋白,包括15 种差异表达蛋白和25 种特异表达蛋白。将驴初乳与常乳中的差异蛋白进行生物信息学分析发现,差异蛋白主要参与的细胞组成为细胞外泌体、胞外囊泡、胞外细胞器等;主要参与的生物过程为对外部刺激的反应过程、细胞增殖过程、血管形态生成过程等;主要参与的分子功能为金属离子结合、阳离子结合、钙离子结合等。此外,这些差异蛋白主要参与补体和凝血级联,为生成IgA而形成的肠道免疫网络等代谢通路。利用蛋白质网络互作分析发现,差异蛋白中存在具有高连接度的关键乳脂球膜蛋白因子。本研究有助了解驴乳脂球膜蛋白的生物学特性,并为驴乳中乳脂球膜蛋白的营养学研究及相关配方乳粉的研发提供理论基础。     

Quantitative analysis of differentially expressed milk fat globule membrane proteins between donkey and bovine colostrum based on high-performance liquid chromatography with tandem mass spectrometry proteomics

This study was designed to provide novel insights into milk fat globule membrane (MFGM) proteins in donkey colostrum (DC) and bovine colostrum (BC) using quantitative proteomics. In total, 179 (DC) and 195 (BC) MFGM proteins were characterized, including 71 shared, 108 DC-specific, and 124 BC-specific proteins. Fifty-one shared proteins were selected as differentially expressed MFGM proteins, including 21 upregulated and 30 downregulated proteins in DC. Gene ontology analysis showed that these proteins were mainly enriched in cellular components, including the extracellular exosome, extracellular space, and plasma membrane. Additionally, they were further involved in metabolic pathways, including cholesterol metabolism, the peroxisome proliferator-activated receptor signaling pathway, and purine metabolism. Furthermore, several key protein factors with high connectivity were identified via protein–protein interaction analysis. These results provide more comprehensive knowledge of differences in the biological properties of MFGM proteins in DC and BC as well as pave the way for future studies of the nutritional and functional requirements of these important ingredients toward the development of dairy products based on multiple milk sources.     

Characterization of bovine neutrophil gelatinase-associated lipocalin

A protein of relative molecular mass of approximately 25,000 was purified from bovine colostrum by cation-exchange and size-exclusion chromatography. The N-terminus of the protein matched the sequence predicted by the National Center for Biotechnology Information for the bovine homolog of human neutrophil gelatinase-associated lipocalin, a glycoprotein of relative molecular mass 25,000 belonging to the family of lipocalins. The protein was further designated as bovine neutrophil gelatinase-associated lipocalin (bNGAL). Sodium dodecyl sulfate-PAGE of enzymically deglycosylated bNGAL indicated that the intact protein bears one N-linked glycan. Monosaccharide and mass spectrometric analyses of released N-linked carbohydrates revealed the presences of complex- and hybrid-type glycans, with galactose substituted with N-acetylgalactosamine. This substitution is typical for glycoproteins expressed in the bovine mammary gland. A specific ELISA revealed bNGAL concentrations in plasma and mature milk of about 0.05 and 1 μg/mL, respectively, whereas values as high as 51 μg/mL were measured in colostrum. Thus, we have isolated and characterized a novel bovine (milk) protein that is a new member of the lipocalin family.     

Bovine milk contains microRNA and messenger RNA that are stable under degradative conditions

We previously reported that microRNA (miRNA) is present in human breast milk. Recently, other groups have reported that bovine milk also contains miRNA; however, these reports are few. We therefore investigated bovine milk miRNA using microarray and quantitative PCR analyses to identify the differences between colostrum and mature milk. The RNA concentration in a colostrum whey fraction was higher than that in a mature milk whey fraction. In total, 102 miRNA were detected in bovine milk by microarray analysis (100 in colostrum and 53 in mature milk; 51 were common to both). Among these miRNA, we selected several immune- and development-related miRNA, including miR-15b, miR-27b, miR-34a, miR-106b, miR-130a, miR-155, and miR-223. These miRNA were detected in bovine milk by quantitative PCR, and each of these miRNA was significantly more highly expressed in colostrum than in mature milk. We also confirmed the presence of some mRNA in bovine milk. Nevertheless, synthesized miRNA spiked in the raw milk whey were degraded, and naturally existing miRNA and mRNA in raw milk were resistant to acidic conditions and RNase treatment. The RNA molecules in milk were stable. We also detected miRNA and mRNA in infant formulas purchased from Japanese markets. It is still unknown whether milk-derived RNA molecules play biological roles in infants; however, if milk-derived RNA do show functions in infants, our data will help guide future studies.     

Distribution of bovine milk sialoglycoconjugates during lactation

The sialoglycoconjugate content of human milk has been extensively studied. However, little attention has been paid to the changes occurring in these compounds in bovine milk during lactation. Since sialoglycoconjugates are very abundant in milk from the early stages of lactation, they have been suggested to be important for the nutrition of the newborn during the first months of life. The distribution of sialoglycoconjugates (expressed as glycoconjugate-bound sialic acid) from four different stages of lactation (colostrum, transitional, mature, and late-lactation milks) was investigated in four Spanish-Brown cows. All the fractions studied (total sialic acids, glycoproteins, oligosaccharides, casein, and gangliosides) showed a similar trend. We found the highest values in the colostrum, these decreasing in transitional and mature milks and increasing again in late-lactation milk. We also found a selective change in the relative contents of glycoprotein- and oligosaccharide-bound sialic acids. In mature milk, the latter increased up to 80% (59% in colostrum) and the former decreased to 3.9% (35.3% in colostrum). It would appear that the decrease in oligosaccharide-bound sialic acid is compensated by the increase in glycoprotein-bound sialic acid. From these results, it is deduced that newborn infants or calves fed with infant formulas or milk replacers, respectively, should be supplemented with sialoglycoconjugates to approximate the composition of human and cow milk as far as is practicable.