Recent advances in mass spectrometry-based proteomics of gastric cancer

The last decade has witnessed remarkable technological advances in mass spectrometry-based proteomics. The development of proteomics techniques has enabled the reliable analysis of complex proteomes, leading to the identification and quantification of thousands of proteins in gastric cancer cells, tissues, and sera. This quantitative information has been used to profile the anomalies in gastric cancer and provide insights into the pathogenic mechanism of the disease. In this review, we mainly focus on the advances in mass spectrometry and quantitative proteomics that were achieved in the last five years and how these up-andcoming technologies are employed to track biochemical changes in gastric cancer cells. We conclude by presenting a perspective on quantitative proteomics and its future applications in the clinic and translational gastric cancer research.

iTRAQ-based quantitative proteome characterization of wheat grains during filling stages

Using isobaric tags for relative and absolute quantification （iTRAQ） and associated analytic technologies, we have cataloged and compared 7 069 unique wheat proteins expressed during four substages of the filling stage. Among them, 859 are differentially expressed, showing at least a 2-fold difference in concentration across substages. Differentially expressed proteins （DEPs） includind high-molecular weight giutenin subunit （W5AIU1）, low-molecular weight glutenin subunit （QSW3V4）, gliadin/avenin-like seed protein （D2KFG9）, and avenin-like protein （W5DVL2）, all of which have previously been identified as important for nutritional quality and bread-making properties, and all of which were found to increase at the latter stages of development. We have applied statistical techniques to group the proteins into hierarchical clusters, and have consulted databases to infer functional and other relationships among the identified proteins.

Proteomic Analysis of Chrysanthemum Lateral Buds after Removing Apical Dominance Based on Label-Free Technology

Studying the genetic basis and regulatory mechanism of chrysanthemum lateral bud outgrowth is of great significance for reduction the production cost of cut chrysanthemum.To clarify the molecular basis of lateral bud elongation after removal of apical dominance in chrysanthemum,label-free quantification analysis was used to analyze the proteome changes after apical bud removal.Quantitative real-time PCR(qPCR)was used to analyze the changes in the expression of three plant hormone-related genes.A total of 440 differentially expressed proteins were successfully identified at three time points during the lateral bud elongation.The number of differentially expressed proteins in the three stages(24 h/0 h,48 h/0 h,48 h/24 h)were 219,332,and 97,respectively.The difference in expressed proteins in the three comparison stages mainly involves RNA processing and modification;translation,ribosomal structure and biogenesis;Posttranslational modification,protein turnover,and chaperones.Path analysis showed that there was various physiological activities in the process of lateral bud dormancy breaking and elongation,which involved energy metabolism,biosynthesis,signal transduction and stress response in the growth process of lateral buds.qPCR indicated that the expression of cytokinin synthesis related gene was significantly increased after the removal of apical dominance,while the expression of strigolactones synthesis related gene experiences a dramatic fall to promote the development of the lateral buds.However,there was a drop before a slight increase in the expression of the auxin synthesis related gene,which was mainly due to the removal of apical dominance that led to the loss of indoleacetic acid in the main stem.However,with formation of the new apical source,indoleacetic acid can be released again.

不同养殖模式大黄鱼非标记定量差异蛋白组学分析

通过非标记定量蛋白质组学技术研究不同养殖模式大黄鱼的蛋白质差异。选取普通网箱养殖大黄鱼和深水网箱养殖大黄鱼,提取肌肉总蛋白,通过高效液相色谱-质谱联用技术进行鉴定、分析。质谱数据使用MaxQuant软件将峰信号转化为肽段/蛋白的表达矩阵数据,然后使用Perseus软件可视化展示数据。不同养殖模式大黄鱼蛋白质进行Student’s t检验,筛选出差异表达蛋白后并对其进行基因功能分类、代谢通路富集和蛋白质相互作用网络分析,以P<0.05为差异有统计学意义。通过数据库比对获得6077条多肽,分别对应于1232个蛋白,其中130个蛋白在大黄鱼间存在显著差异表达,76个上调蛋白,54个下调蛋白,其中上调倍数最高的属于TCP伴侣蛋白,下调倍数最高的属于α-1(I)链状胶原蛋白。生物信息学分析发现,不同养殖模式大黄鱼蛋白质的差异主要在于热休克蛋白、伴侣蛋白、球蛋白及胶原蛋白等,而不同养殖模式中环境温度可能是导致大黄鱼蛋白质显著差异表达的主要因素。

Effect of Drought Stress on Proteome of Maize Grain during Grain Filling

Based on isobaric tags for relative and absolute quantification(iTRAQ)technology,the proteome of grains of a maize cultivar Huangzao 4 under drought stress at grain filling stage was analyzed.The results show that under drought stress,438 proteins were differentially expressed in the maize grains during grain filling.Among them,200 were up-regulated and 238 were down-regulated.The gene ontology(GO)analysis shows that the biological processes in which differential proteins are more involved are cellular processes,metabolic processes and single biological processes;proteins in the cell component category are mainly distributed in cells,cell parts and organelles;and the proteins the molecular function category mainly possess catalytic activity and binding function.Differentially expressed proteins classified by COG are mainly involved in protein post-translational modification and transport,molecular chaperones,general functional genes,translation,ribosomal structure,biosynthesis,energy production and transformation,carbohydrate transport and metabolism,amino acid transport and metabolism,etc.The subcellular structure of the differentially expressed proteins is mainly located in the cell chloroplast and cytosol.The proportions are 35.01%and 30.21%respectively.KEGG metabolic pathway enrichment analysis shows that the differentially expressed proteins are mostly involved in antibiotic biosynthesis,microbial metabolism in different environments,and endoplasmic reticulum protein processing;the metabolic pathways with higher enrichment are the carbon fixation pathway and estrogen signaling pathway of prokaryotes;and the higher enrichment and greater significance are in the tricarboxylic acid cycle,carbon fixation of photosynthetic organisms and proteasome.The results of this study preliminarily reveal the adaptive mechanism of maize grains in response to drought stress during grain filling,providing a theoretical reference for maize drought-resistant molecular breeding.

Gehua Jiejiu Dizhi decoction(葛花解酒涤脂汤)ameliorates alcoholic fatty liver in mice by regulating lipid and bile acid metabolism and with exertion of antioxidant stress based on 4DLabel-free quantitative proteomic study

OBJECTIVE:To analyze the effect and molecular mechanism of Gehua Jiejiu Dizhi decoction(葛花解酒涤脂汤,GJDD)on alcoholic fatty live disease(AFLD)by using proteomic methods.METHODS:The male C57BL/6J mouse were randomly divided into four groups:control group,model group,GJDD group and resveratrol group.After the AFLD model was successfully prepared by intragastric administration of alcohol once on the basis of the Lieber-DeCarli classical method,the GJDD group and resveratrol group were intragastrically administered with GJDD(4900 mg/kg)and resveratrol(400 mg/kg)respectively,once a day for 9 d.The fat deposition of liver tissue was observed and evaluated by oil red O(ORO)staining.4DLabel-free quantitative proteome method was used to determine and quantify the protein expression in liver tissue of each experimental group.The differentially expressed proteins were screened according to protein expression differential multiples,and then analyzed by Gene ontology classification and Kyoto Encyclopedia of Genes and Genomes pathway enrichment.Finally,expression validation of the differentially co-expressed proteins from control group,model group and GJDD group were verified by targeted proteomics quantification techniques.RESULTS:In semiquantitative analyses of ORO,all kinds of steatosis(ToS,MaS,and MiS)were evaluated higher in AFLD mice compared to those in GJDD or resveratroltreated mice.4DLabel-free proteomics analysis results showed that a total of 4513 proteins were identified,of which 3763 proteins were quantified and 946 differentially expressed proteins were screened.Compared with the control group,145 proteins were up-regulated and 148 proteins were down-regulated in the liver tissue of model group.In addition,compared with the model group,92 proteins were up-regulated and 135 proteins were downregulated in the liver tissue of the GJDD group.15 differentially co-expressed proteins were found between every two groups(model group vs control group,GJDD group vs model group and GJDD group vs control group),which were involved in many biological processes.Among them,11 differentially co-expressed key proteins(Aox3,H1-5,Fabp5,Ces3a,Nudt7,Serpinb1a,Fkbp11,Rpl22l1,Keg1,Acss2 and Slco1a1)were further identified by targeted proteomic quantitative technology and their expression patterns were consistent with the results of 4D label-free proteomic analysis.CONCLUSIONS:Our study provided proteomics-based evidence that GJDD alleviated AFLD by modulating liver protein expression,likely through the modulation of lipid metabolism,bile acid metabolism and with exertion of antioxidant stress.

全血和血斑蛋白质组学差异分析

目的 研究外周血形成干燥血斑后蛋白质水平的变化。方法 应用液相色谱-串联质谱法(LCMS/MS)和非标记定量(label-free quantification,LFQ)策略进行全血和血斑的蛋白质检测,分别采用R 4.2.1软件limma包和edgeR包筛选差异蛋白质,再进行生物学功能、信号通路和亚细胞定位分析。结果 全血和血斑中分别检测到623个和596个蛋白质,其中31个蛋白质的定量差异具有统计学意义,包括10个丰度在血斑中上调和21个丰度在血斑中下调的蛋白质。结论 全血和血斑中的蛋白质丰度高度相关,两者间蛋白质丰度的改变可能与细胞内源性蛋白和结构蛋白的变化相关。应用蛋白质组学技术可以辅助蛋白质生物标志物的筛选和鉴定,为法医学研究引入新型生物标志物。

iTRAQ技术及其在蛋白质组学中的应用

近年来随着蛋白质组学的迅速发展,其相应的方法学研究也取得了巨大的进步,一系列新技术融入了蛋白质组学研究中,极大地促进了这门学科的发展.相对和绝对定量同位素标记(iTRAQ)技术与高度敏感性和准确性的串联质谱及多维液相色谱联用技术已成为蛋白质定性和定量研究的主要工具之一.该技术可对复杂样本、细胞器、细胞裂解液等样本进行相对和绝对定量研究,具有较好的定量效果、较高的重复性.由于其能够同时对多达8种样品进行标记分析,故在生命科学的各个领域得到了广泛的应用.本文对iTRAQ的原理、实验流程、优缺点及近几年的应用进展进行综述.

蛋白质质谱分析的无标记定量算法研究进展

作为发现疾病相关生物标志物的重要途径,定量研究已成为蛋白质组学的热点问题.随着实验方法的发展和改进,定量数据处理算法也在不断更新和完善.将现有的无标记定量方法归纳为需要/不需要鉴定结果两类方法,分析比较了两类方法的异同及优缺点,详细讨论了所涉及的主要算法,总结了一些常用的无标记定量软件及对应的网络资源.展望了无标记定量数据分析的未来研究方向.

食物过敏原蛋白的定量检测方法

食物过敏现象的日益严重凸显食物过敏原蛋白定量检测的重要性。目前对食物过敏原蛋白进行定量检测的方法多是基于单个蛋白的间接测定。随着质谱分析方法的进步,定量蛋白质组学技术已开始并将广泛应用于复杂食品混合物中微量过敏原的直接定量检测。本文分别对基于单个蛋白和蛋白质组学的食物过敏原蛋白的定量测定方法进行综述。

定量蛋白质组学揭示三角鲂和团头鲂响应嗜水气单胞菌侵染机制变化

细菌性败血症对鱼类危害严重,为探究鲂属鱼类不同抗性品种响应病原菌嗜水气单胞菌的抗性机制,以三角鲂(高抗)和团头鲂(易感)肝组织为材料,采用非标记定量蛋白质组学技术分析其在嗜水气单胞菌侵染胁迫后3、10和24h与对照组0h(未感染)肝组织蛋白质组的变化,对差异表达蛋白质进行质谱鉴定、相对定量和生物信息学分析。结果显示:三角鲂和团头鲂在嗜水气单胞菌侵染下,分别有46个和29个蛋白质在侵染后各时期的表达丰度相比对照都发生了显著变化。通过基因本体(gene ontology,GO)功能注释发现,相对于团头鲂,三角鲂中氧化还原蛋白质比例从7%增加到13%,且发现一类新的β-免疫球蛋白(β-globin)在各时期分别上调表达了1.64、3.44和1.93倍。生物信息学分析进一步表明,三角鲂在响应嗜水气单胞菌侵染过程中可能特异性地启动了包括戊糖磷酸途径、脂肪酸代谢、亮氨酸代谢途径和β-globin高表达的协同抗性机制,从而抵御病原菌入侵。该研究成果为后续深入揭示鱼病互作分子机制及鲂属鱼类抗病新品种选育提供了重要的前期理论研究基础。

蛋白质组学技术前沿进展

蛋白质组学是以生物体系整体蛋白质为研究对象的新的研究领域,已经成为后基因时代中生命科学最重要研究方向之一。近年来,蛋白质组学研究取得了令人鼓舞的进展,一系列新技术与新方法得到了快速的发展。本文总结了2013年以来蛋白质组学研究的有关新技术,并对其发展进行了展望。

蛋白质组学定量的技术与方法研究进展

蛋白质组学是一门在整体水平上研究细胞内蛋白质组成及其活动规律的新兴学科。定量蛋白质组学是指通过某种方法或技术,对生物样品(细胞、组织或体液等)在某些过程中蛋白质的含量进行比较分析。近几年来,定量蛋白质组的技术发展很快,稳定同位素标记技术的提出,为准确定量在细胞或组织体系中发挥重要功能的低丰度蛋白质提供了一个理想的方法。本文综述了蛋白质组定量分析技术及其最新的研究进展。

邻苯二甲酸二乙基己酯(DEHP)毒理与健康效应研究进展

邻苯二甲酸二乙基己酯(DEHP)作为重要的聚氯乙烯塑料增塑剂在工业上应用广泛。目前,DEHP在海洋、大气、饮用水及动植物体内均被不同程度检出,对生态环境造成日益严重的污染。结合国内外毒理学研究成果,概述DEHP的人体暴露及代谢途径;并从肝脏、心脏、生殖发育系统和呼吸系统等方面详细探讨DEHP对动物健康的危害及其可能的毒性机制,以及对人体健康潜在的影响;在此基础上,对目前存在的问题及进一步研究的方向进行了探讨和展望。

二维电泳和iTRAQ的实验比较

对蛋白组学中的二维电泳和iTRAQ两种技术进行比较研究,为神经科学的蛋白组学研究提供选择参考。取生后1～3d C57Bl/6小鼠背根神经节进行培养,对照组加DMSO,实验组加JNJ460。分别用二维电泳和iTRAQ两种技术检测背根神经节细胞蛋白的变化,用4700 TOF/TOF串联飞行质谱仪检测变化蛋白的肽片段质量,用GPS软件检索Mascot蛋白质数据库,鉴定出匹配的蛋白。利用二维电泳技术观察到4种表达上调的蛋白,2种是结构蛋白,2种是信号转导蛋白。利用iTRAQ技术观察到22种表达上调的蛋白,其中10种是结构蛋白,3种是信号蛋白,2种是代谢蛋白,2种是降解蛋白化,2种是细胞基质蛋白及3种其它蛋白。利用二维电泳技术观察到发生表达变化的蛋白都位于胞浆内,而利用iTRAQ技术观察到的表达变化的蛋白有胞浆蛋白外,还有线粒体蛋白、膜蛋白和核蛋白。同时,利用二维电泳技术观察到的蛋白变化都在2倍以上,而利用iTRAQ技术计算出的蛋白变化在1.3至1.6倍之间。另,iTRAQ技术在鉴定大分子和小分子蛋白方面也有优势。结果表明iTRAQ技术比二维电泳技术能发现更多数量和更多种类的蛋白,但在比较胞浆蛋白的变化及蛋白量的变化方面二维电泳技术有一定的长处,对iTRAQ的实验结果有互补作用。

氟西汀作用于慢性温和不可预见性应激大鼠海马组织前后的差异蛋白质组学研究

目的:采用蛋白组学方法研究氟西汀对慢性温和不可预见性应激(chronic unpredictable mild stress,CUMS)大鼠海马组织蛋白质表达的影响,以探讨氟西汀抗抑郁的作用机制。方法:采用CUMS建立大鼠抑郁模型。造模完成后,氟西汀组给予氟西汀(3 mg/kg)灌胃给药,每天1次,连续21 d。行为学实验检测大鼠抑郁行为的变化。提取的大鼠海马组织蛋白质样品经同位素标记相对和绝对定量标记、强阳离子柱分离、反相液相色谱分离、质谱分析,获取的串联质谱数据通过Protein.pilot 3.0软件鉴定蛋白质,运用生物信息学分析差异表达的蛋白质功能。结果:共鉴定出5 109个蛋白质,41个差异表达的蛋白质,其中36个蛋白质表达上调,5个蛋白质表达下调,涉及氧化还原、信号传导、合成代谢、对外界刺激压力的反应等过程。结论:同时筛选到了四个可能与氟西汀抗抑郁作用密切相关的差异蛋白,分别是Neuronal calcium sensor 1(NCS-1)、Receptor-type tyrosine-protein phosphatase zeta、Neurocan core protein和Sodium-and chloride-dependent GABA transporter,但这些蛋白在抑郁症的发生、发展过程中的作用尚未完全清楚,需进一步深入研究。

选择性反应监测技术在蛋白质组学研究中的进展及应用

选择性反应监测(SRM)技术作为一种重要的定向蛋白质分析技术,通过选择性检测特定母离子和子离子来排除非目标组分的干扰,增强了检测灵敏度和定量准确度,具有选择性高、重复性好、灵敏度高、动态范围宽等优点,已被广泛应用于定量蛋白质组学研究,在生命科学领域发挥着至关重要的作用。本文从分析通量、检测灵敏度、定量方法以及相关软件资源4个方面,对近期SRM技术的研究进展进行了综述。然后,对SRM技术在蛋白质组学研究包括生物标志物验证、蛋白质翻译后修饰研究、生物工程以及信号通路分析等领域中的应用进行了概述。最后,本文对SRM技术的应用以及发展前景进行了展望。

癌症差异蛋白质组学研究中样品分离和鉴定分析技术

随着人类基因组测序的完成,癌症研究的重点从基因组学转移到蛋白质组学研究中。癌症研究中的差异蛋白质组学技术也飞速发展,包括癌症样品制备、分离,蛋白质鉴定分析、蛋白质组定量研究和翻译后修饰研究等。这些技术极大地推动了与癌症相关的差异蛋白质组学研究,使蛋白质组学在癌症早期诊断、治疗,监测以及发现新药物治疗靶标方面发挥更大的作用。本文主要综述了近年来癌症差异蛋白质组学研究中样品分离和鉴定分析技术。

应用iTRAQ多重化学标记串联质谱技术筛选晚期卵巢浆液性腺癌组织中卡铂耐药相关蛋白

目的利用同位素标记相对和绝对定量(iTRAQ)技术筛选晚期卵巢癌组织中卡铂耐药相关差异表达蛋白,为临床个体化治疗奠定实验基础。方法收集Ⅲ期低分化卵巢浆液性腺癌标本并通过ATP-TCA药敏试验检测卡铂敏感度。取卡铂敏感和耐药标本各15例,iTRAQ试剂标记,被标记的肽段进行高效液相色谱(HPLC)分离及质谱检测(MS)。结果共鉴定出iTRAQ标记定量信息有755个显著差异表达的蛋白。卡铂耐药组与卡铂敏感组相比,上调1.2倍以上的蛋白429个;下调0.83倍以下的蛋白326个。上调蛋白中有osteopontin(OPN)、clusterin(CLU)、5'-nucleotidase(CD73)和tissue inhititor of metalloproteinases 1(TIMP1)等18个蛋白与肿瘤恶性行为及化疗耐药相关。结论应用iTRAQ技术能筛选出多种与晚期卵巢癌卡铂耐药相关的差异表达蛋白,该技术对于肿瘤组织蛋白质组学的研究有很好的应用前景。

同位素标记相对和绝对定量蛋白组技术研究进展

近年来定量蛋白组学技术迅速发展,目前常用的有双向荧光差异凝胶电泳、同位素亲和标记、15N同位素标记、同位素标记相对和绝对定量和细胞培养条件下稳定同位素标记技术等。同位素标记相对和绝对定量技术以其高通量、高灵敏度、高重复性、高动态检测限和能对各种复杂样品进行相对和绝对定量研究等优势而备受研究者青睐,目前已发展到在同一实验中分析8组样品,增加了实验设计的灵活性。我们就同位素标记相对和绝对定量技术在定量蛋白组史中的地位作用、研究策略,以及在病毒致病机制研究和医药临床相关问题中的应用做简要综述。