蛋白质组学在人乳蛋白质研究中的应用

人乳是新生儿最理想的天然食物，蛋白质是人乳中最主要的营养成分之一。随着蛋白质组学技术的发展，利用蛋白质组学的方法研究人乳蛋白质也取得了一些研究成果。本文综述了近年来蛋白质组学技术在人乳蛋白质研究中的应用，分别从人乳蛋白质的组成研究、动态变化、人乳与其他来源乳汁的蛋白质差异比较、人乳磷酸化蛋白和糖基化蛋白研究、人乳内源肽的研究及人乳蛋白与疾病等几个方面进行了阐述。蛋白质组学技术使人乳蛋白质的研究进入了微量营养研究的时代，人乳蛋白质组学的研究成果将为母婴健康提供更好的保障。     

蛋白质水解阶段对氨基酸组成分析的影响

水解是蛋白质氨基酸组成分析的重要步骤，水解质量的好坏直接影响到分析结果的正确与否，蛋白质的水解受到很多因素的影响，如温度，时间、水解试剂，添加剂，水解方法等，本文对近10年来蛋白质组成分析中中有关蛋白质水解问题的研究进进行了评述，对最近几年出现的一些新的水解方法如酸性水解中的气相水解，微波辐射水解，膜上蛋白质和印迹水解，聚丙烯酰安凝胶中蛋白质的水解，敏感氨基酸的水解以及水解过程中影响氨基酸外消旋化的因素等作了简要的介绍。     

蛋白质水解液中氨基酸组成的定量分析

以氯甲酸－9－芴基甲酯（FMOC－Cl）为柱前衍生化试剂在液相色谱上分离17种常见氨基酸的衍生物并测定其摩尔吸光系数。用绝对分析法和标准曲线法对标准氨基酸溶液进行了定量分析，除组氨酸外两种方法测定结果的相对偏差为5．00％以内。对氨基酸的稳定性进行了模拟水解实验，发现在110℃/24h的水解条件下，丝氨酸等不稳定氨基酸的降解较在150℃/1.5h条件下小。用绝对分析法测定测定了牛胰岛素不同水解条件下水解液中氨基酸的含量，大部分氨基酸的相对收率都接近100％。     

医用乳胶手套中水溶性蛋白质的含量分析

本文采用改进的Lowry法对医用乳胶手套中水溶性蛋白质析出量进行了测定,并探讨了乳胶手套中水溶性蛋白质析出量与提取时间的关系.统计学结果显示:2 h、4 h和8 h组所测得的蛋白质析出量均值分别为124.14±29.09 μg/g、 183.58 ±33.22 μg/g和190.58±29.97 μg/g.通过t检验,2 h组和4 h组相比较,2 h组和8 h组相比较,差异均有高度统计学意义(p=0.000);4 h组和8 h组相比较,差异无统计学意义(p=0.563).     

分析蛋白质-蛋白质相互作用界面的新方法

疏水性和氢键是蛋白质-蛋白质相互作用中的主要因素.提出一种新的计算方法，从蛋白受体的结合部位推导出蛋白配体相应部位应该具有的疏水性质和氢键性质.应用这种方法可以很容易地找出影响相互作用的关键残基，并且将界面的这两种特征用图形软件显示出来.在应用到实际蛋白-蛋白相互作用中时，发现它的用途并不限于此.它可以作为研究蛋白相互作用的一个基本工具.     

中性人乳寡糖的分离及结构鉴定

以脱脂、除蛋白后的人乳为原料,联合运用阴离子交换色谱(AEC)、凝胶渗透色谱(GPC)和多孔石墨化碳色谱(PGC)等多种分离纯化技术制备了18种中性人乳寡糖,并采用电喷雾碰撞诱导串联质谱(ESI-CIDMS/MS)技术对其结构进行了鉴定.结果表明,所制备的中性人乳寡糖主要为三至九糖,含有半乳糖(Gal)、葡萄糖(Glc)、N-乙酰葡萄糖胺(GlcNAc)和岩藻糖(Fuc).所有寡糖的还原端均含有乳糖核心(Galβ1-4Glc),非还原端均含有乳糖胺(Galβ1-3/4GlcNAc),并且Fuc以α1-2,α1-3和α1-4连接于主链的Gal,Glc和GlcNAc上.     

蛋白质芯片及其分析应用新进展

蛋白质芯片是一种快速、高效、高通量的蛋白质组研究新技术。目前,它已成为人们研究的热点之一。本文就近年来蛋白质芯片及其分析应用新进展做一简要的评述。     

蛋白质-蛋白质分子对接中打分函数研究进展

分子对接是研究分子间相互作用与识别的有效方法.其中,用于近天然构象挑选的打分函数的合理设计对于对接中复合物结构的成功预测至关重要.本文回顾了蛋白质-蛋白质分子对接组合打分函数中一些主要打分项,包括几何互补项、界面接触面积、范德华相互作用能、静电相互作用能以及统计成对偏好势等打分项的计算方法.结合本研究小组的工作,介绍了目前普遍使用的打分方案以及利用与结合位点有关的信息进行结构筛选的几种策略,比较并总结了常用打分函数的特点.最后,分析并指出了当前蛋白质-蛋白质对接打分函数所存在的主要问题,并对未来的工作进行了展望.     

生命科学中的分析化学--蛋白质组分析

概述了分析化学在生命科学中的重要作用和蛋白质组分析的现状与展望。介绍了生物试样的制备、双向凝胶电泳分离蛋白质以及生物质谱鉴定蛋白质等蛋白质组分析的主要技术平台。     

奶制品中蛋白质测定的毛细管电泳法研究

本文利用毛细管电泳法研究了奶制品中蛋白成分的分离与测定。采用柠檬酸缓冲体系,对牛奶中的α-乳白蛋白(-αLa)、β-乳球蛋白(-βLg)、α-酷蛋白(-αCN)、β-酪蛋白(-βCN)、酪蛋白(-κCN)五种蛋白进行了很好的分离,其迁移时间和峰面积的相对标准偏差分别小于0.2%和5%;检出限分别为0.01、0.03、0.02、0.02、0.02 mg/mL;加标回收率范围为84%~103%。应用该法测定了多种奶制品中的蛋白质含量。本法简便、快速、准确,为牛奶及奶制品质量的监控提供了一种新的手段。     

Separation of Proteins in Human Milk

Abstract

A separation of proteins in human milk whey using high performance ion exchange chromatography is reported. Three chromatographic peaks were identified on the basis of retention times as bile salt-stimulated lipase (BSSL), lactoferrin, and α-lactalbumin. Implications for the purification of BSSL are discussed and suggestions for future purification procedures are made.     

Amino-Acid Composition of Camel's Milk and Shubat

A comparative analysis of the amino-acid content of camel's milk and shubat from the Aral region and Kazakhstan was carried out. The particular amino-acid composition and different quantitative content were determined.     

反相高效液相色谱法测定牛奶中的主要蛋白质

建立了测定牛奶中的主要蛋白质的反相高效液相色谱法,对前处理方法进行了优化,采用Agilent Zorbax 300SB-C8(250 mm×4.6 mm,5 μm)色谱柱,三氟乙酸-水-乙腈流动相梯度洗脱,214 nm检测,柱温45 ℃,外标法定量.测定κ-CN, αs2-CN, αs1-CN, β-CN, Whey和Igg的线性关系良好,相关系数均大于0 999,加标回收率在74.8%～132.5%之间.大豆蛋白质不影响分离与检测.采用本方法分析了9种牛奶样品中上述蛋白质的含量与总量,结果表明,本方法测定结果准确可靠.不同样品中4种酪蛋白与乳清蛋白的比例基本接近,但是不同品牌之间存在一定差异.     

母乳中甲硝唑、替硝唑测定及药代动力学

采用高效液相色谱法测定哺乳妇女单剂量静滴甲硝唑 ( 2 0mg/kg ,n =8)、替硝唑 ( 1 3mg/kg ,n =7)乳汁药物浓度 ,色谱柱为 pecoshereC1 8( 3μm ,3.3cm× 4.6mm) ,氯仿提取乳汁中药物 ,其平均回收率大于 88.8% .乳汁中甲硝唑与替硝唑药代动力学参数tmax,Cmax,t1 / 2Ke分别为 1 .7± 1 .0h ,2 0 .1 0± 4.95μg/mL ,6.4± 3.3h和 1 .3±0 .6h ,1 7.2 3± 3.1 2 μg/mL ,1 1 .0± 3.5h .     

An Integrative Approach of an In Vitro Measurement of the Digestibility of Triacylglycerols of Human Milk

Several studies have been published regarding the effect of different factors on the digestion of milk lipids, considering their natural structural arrangement as milk fat globules and the efficiency of the digestive enzymes in the lipolysis of such complex structures. During digestion, the lipolytic products are dispersed in vesicles and micelles, which are the source for absorption of digested lipids. Therefore, it is necessary to consider the isolation of the micellar phase from the digesta to appropriately determine the amounts and classes of lipids which are bioaccessible. This study presents an integrative approach that included an isolation procedure to separate the micellar fraction from undigested and non-micellar parts, and the distribution of digested milk lipids in micelles determined directly through chromatographic techniques. Four groups of five full term mothers donated colostrum or mature milk. Two sets of samples were analyzed directly (raw), and two sets were pasteurized and then analyzed. Our data revealed that the profile of digested milk lipids is different depending on the lactation period and processing stage, while the carbon atom number distribution of the digested triacylglycerols in the micellar fraction provides a substantial information regarding the acylglycerols species that are less available for absorption.     

电喷雾多级串联质谱技术区分人乳寡糖异构体

利用四极杆-飞行时间(Q-TOF)电喷雾多级串联质谱(ESI-MS/MS)与诱导碰撞解离技术(C ID)在负离子模式下选择合适碰撞能量对几组互为异构体的人乳寡糖进行了分析,并对其碎裂机理进行了探讨。结果表明:在二级质谱中碰撞能量范围为15~30 eV时,寡糖异构体产生其特征碎片离子,据此可以快速灵敏地区分寡糖异构体。     

1H NMR Metabolomics of Chinese Human Milk at Different Stages of Lactation among Secretors and Non-Secretors

Human milk is an intricate, bioactive food promoting infant health. We studied the composition of human milk samples collected over an 8-month lactation using ¹H NMR metabolomics. A total of 72 human breast milk samples were collected from ten Chinese mothers at eight different time points. The concentrations of ten human milk oligosaccharides (HMOs), fucose and lactose were quantified. Six of the mothers were classified as Lewis-positive secretors (Se⁺Le⁺) and four as Lewis-positive non-secretors (Se⁻Le⁺) based on the levels of 2′-fucosyllactose (2′-FL), lacto-N-fucopentaose (LNFP) II, lactodifucotetraose (LDFT) and lacto-N-neotetraose (LNnT). Acetate, citrate, short/medium-chain fatty acids, glutamine and urea showed a time-dependent trend in relation to the stage of lactation. The concentrations of 2′-FL, 3-FL (3-fucosyllactose), 3′-SL (3′-sialyllactose), LDFT, LNFP I, LNFP II, LNFP III, LNnT, LNT (lacto-N-tetraose), and fucose were statistically different between secretors and non-secretors. A temporal difference of approximately 1–2 months between the development of non-secretor and secretor HMO profiles was shown. The results highlighted the importance of long-term breastfeeding, especially among non-secretors.     

Analysis of Free Amino Acids and Protein Contents of Mature Human Milk from Turkish Mothers

Abstract

This study was designed to evaluate free amino acid (FAA) composition and total protein in mature human milk from Turkish mothers. Free amino acid concentrations in mature human milk were determined in all subjects using a high‐performance liquid chromatography (HPLC) with postcolumn derivatization system, with a fluorescence detector. Total protein content was determined by the classical biuret method. Total protein concentration was found to be 1.3±0.4 mg/dl. Glutamic asid plus glutamine is the most abundant amino acid (1275 µmol/L), followed by taurine (353 µmol/L) and alanine (261 µmol/L). Glutamic acid plus glutamine accounts for the most free amino acids in mature human milk and their sum represents 40% of total FAA. On the other hand, some amino acid derivatives such as citrulline, ethanolamine, ammonium, ornithine, ortophosphoserine, and phosphoethanolamine, not usually a part of protein, are determined and this fraction represented ～21% of the total FAA in mature human milk in the present study.