质谱分析及其在肺癌分子标志物研究中的应用

常压直接质谱分析技术和质谱成像技术是近年来质谱分析的重要进展,它们在生物样品分析 中具有广泛应用价值。肺癌是最常见的恶性肿瘤之一,早期筛查和诊断是改善其临床诊治现状的重要 途径。本文就质谱分析及其在肺癌分子标志物研究中的应用作一综述。     

MassARRAY质谱分析肺癌多基因突变方法的建立与应用

《中国肿瘤临床》2015年第17期“临床研究与应用”栏目中，来自广东省人民医院吴一龙教授研究团队的《MassARRAY质谱分析肺癌多基因突变方法的建立与应用》一文，通过文献检索并结合国内外研究进展，确定与中国肺癌人群发病、耐药以及转移等密切相关的13个靶基因或相关传导通路基因，对目的基因突变进行筛选并确定99个热点突变，建立MassARRAY质谱分析肺癌多基因突变检测方法，以适应中国肺癌人群的人群多基因同步检测。该文内容具有一定临床应用前景，值得深度阅读。     

新的肺癌血清标志物——甲状腺运载蛋白

背景与目的 肺癌是当今世界上最常见的恶性肿瘤之一,迄今缺乏临床诊断可用的分子标志物.本实验应用SELDI技术寻找肺癌新的血清标志物.方法 对227例血清样品(包括146例肺癌、13例肺炎、28例结核性胸膜炎和40例正常人血清样品)进行蛋白质谱检测.对候选蛋白进行质谱鉴定,并结合免疫共沉淀和ELISA技术筛选出肺癌新的候选标志物.结果 通过Biomarker WizardTM软件分析显示13.78 kDa、13.90 kDa和14.07 kDa的蛋白峰在肺癌病人血清样品中明显低于对照组.通过1-D胶分离,质谱鉴定和免疫沉淀分析显示这3个差异蛋白峰为野生型甲状腺运载蛋白(nativeTTR)和它的两个变体(cysTTR and glutTTR).ELISA和SELDI技术分析上述血清样品均发现TTRs在肺癌血清中的表达下调.结论 采用SELDI技术首次筛选并鉴定出TTRs,表明其可能作为肺癌诊断的候选血清标志物.     

肺癌骨转移血清蛋白质组学研究

目的:应用蛋白质组学双向荧光差异凝胶电泳和质谱技术筛选肺癌骨转移血清标志.方法:收集肺癌伴骨转移、无骨转移各24例血清样本.同组血清等量混合,用除白蛋白试剂盒除去血清中的白蛋白,再经除盐浓缩后,将4组处理后的血清样品用不同的CyDye染料交叉标记后进行双向荧光差异凝胶电泳(2-D DIGE).利用DeCyder软件分析,将肺癌伴骨转移组与肺癌无骨转移组比较的差异蛋白质点行基质辅助激光解析离子化飞行时间质谱(MALDI-TOF-MS)鉴定.结果:双向荧光差异凝胶电泳显示,肺癌骨转移组血清蛋白中有16个斑点,与肺癌无骨转移组相比差异有统计学意义.其中11个蛋白质表达水平显著增加,选取5个蛋白质表达水平显著下降.5个差异蛋白点进行胶内原位酶解、肽质指纹图谱分析,成功得到4个蛋白质肽质指纹图,并查询数据库初步鉴定该4个蛋白质分别为视黄醇结合蛋白-甲状腺素运载蛋白复合物(RBP-TTR)、结合殊蛋白(Hp)、S-亚硝基血红蛋白(SNO-Hb)和de-sArg141 α Hb.结论:双向荧光差异凝胶电泳结合质谱鉴定是血清差异蛋白质组学研究的可靠平台和有力工具.所鉴定出的蛋白质可能与肺癌骨转移的发生、发展有关,有助于肺癌骨转移发病机制的了解.     

应用SELDI—TOF—MS技术于肺癌中医辩证的初步研究

目的 探讨不同证型肺癌患者血清中蛋白质质谱的差异.方法 用WCX2蛋白芯片结合表面增强激光解析电离飞行时间质谱(SELDI-TOF-MS)技术,分别检测12例气阴两虚肺癌和12例气滞血淤肺癌的血清蛋白质谱,筛选出差异表达蛋白质.结果 共检测到113个有效的蛋白质波蜂,其中m/z位于2 000-10 000的波峰有104个,筛选出m/z为3 952.05、3 772.35、4 170.07、3 679.34、3 156.38、4 293.14的6个血清蛋白质.结论 利用SELDI-TOF-MS技术将有可能建立新的中医肺癌辩证模式--蛋白质指纹图谱.     

蛋白质组学及其研究技术在肺癌分子生物标志物研究中的应用

肺癌是全球发病率和死亡率较高的恶性肿瘤。细胞从正常状态转变为肿瘤的过程中，细胞内蛋白质表达谱必然会发生一系列变化，肿瘤分子标记物就是细胞在非正常状态下产生的分子．以高通量结合生物信息学为特点的蛋白质组学分析技术可以从细胞整体水平上检测到这种变化近年来，为了降低肺癌的死亡率并提高其治疗效果，研究人员利用这一新的手段如利用激光捕获显微切割技术和双相凝胶电泳结合基质辅助激光解析电离飞行时间质谱（MALDI-TOF MS）和表面增强激光解析电离色行时间质谱（SELDI—TOF MS）寻找有效的肺癌标记物。本文对蛋白质组学技术在肺癌生物标志物研究中的应用作了综述。     

SELDI-TOF-MS技术在非小细胞肺癌中的应用

表面增强激光解析离子化飞行时间质谱（SELDI-TOF-MS）技术是一种使用特殊的增强表面直接捕获待测样本中的蛋白质分子,然后经激光解析离子化．飞行时间质谱测定被捕获的蛋白质的相对分子量、丰度的新兴技术。目前该项技术已经成功应用于前列腺癌的诊断、子宫良恶性肿瘤的鉴别等方面。SELDI-TOF-MS技术通过寻找肿瘤病人相关的特异性蛋白用于对非小细胞肺癌的早期诊断,指导治疗及预测预后等方面具有很好的应用前景。     

紫杉醇耐药细胞与敏感细胞分泌蛋白差异凝胶电泳分析

背景与目的 紫杉醇是用于肺癌临床一线治疗的作用于微管蛋白的化疗药物,而耐药是影响其疗效的关键因素.本研究应用蛋白组学技术整体比较紫杉醇耐药细胞与敏感细胞分泌蛋白表达谱,以期发现肺癌紫杉醇耐药相关分泌蛋白标志物,为临床选择有针对性的化疗药物提供依据.方法 收集A549亲代及耐药细胞培养液中的蛋白,应用差异凝胶电泳(DIGE)进行分离,Decyder 6.5软件分析后获得的差异蛋白点通过基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)进行鉴定.结果 应用DIGE进行分离后,成功获得了蛋白质组分辨率高、重复性好的双向凝胶电泳图谱,Decyder 6.5软件分析获得差异>2倍以上的蛋白质点共有76个,经质谱鉴定了19种蛋白,鉴定的蛋白涉及代谢酶类、细胞外基质降解酶类、粘附分子、细胞因子和信号转导相关蛋白质.结论 本研究首次应用双向电泳技术整体展示了紫杉醇耐药细胞株及敏感细胞株的分泌蛋白表达谱,并分析、鉴定、筛选出与紫杉醇耐药相关的差异分泌蛋白.该研究为非小细胞肺癌血清耐药标志物的筛选提供了新的方法和候选分子.     

MassARRAY 质谱分析肺癌多基因突变方法的建立与应用*

目的:以MassARRAY质谱iPLEX 分析技术为平台建立适合中国肺癌人群多基因同步检测的方法。方法:通过文献检索并结合国内外研究进展,确定与中国肺癌人群发病、耐药以及转移等密切相关的13个靶基因或相关传导通路基因（EGFR、KRAS、ALK 、FGFR1、FGFR2、FGFR3、PIK3CA、BRAF、PTEN、MET 、ERBB2、AKT 1、STK 11）。 对目的基因突变进行筛选并确定99个热点突变。按MassARRAY突变位点标示方法和引物设计固定格式,引物设计软件在线设计297 条引物（正、反向扩增引物和延伸引物各99条）。 以8 个肺癌细胞系以及6 例肿瘤组织样本建立检测方法,与LungCarta试剂盒对比验证。扩大检测100 例肺癌组织样本,与EGFR 和KRAS直接测序法比较敏感度与特异度。结果:使用本方法检测肺癌细胞系的基因突变,其中1 例肺癌组织新检测到FGFR2 基因突变,其他结果与LungCarta试剂盒一致。与直接测序法相比较灵敏度为100% ,特异度为96.3% 。结论:成功建立MassARRAY质谱分析肺癌多基因突变检测方法,适合中国肺癌人群且具有临床应用前景。      

应用蛋白质芯片技术筛选早中期和晚期肺癌患者血清差异蛋白

 目的　应用蛋白质芯片技术检测早中期和晚期肺癌患者血清中的差异表达蛋白并探讨其临床意义。方法　应用表面增强激光解析电离飞行时间质谱（SELDI-TOF-MS）技术,使用弱阳离子交换（WCX-2）蛋白芯片分别检测两组肺癌患者血清蛋白指纹图谱,采用蛋白质芯片阅读机读取数据,Ciphergen Protein Chip 3.0版本的分析软件采集数据,读取峰信息,进行统计学处理。两组之间蛋白质峰强度的比较采用χ2检验。结果　早中期和晚期肺癌患者血清中存在10个有统计学意义的差异蛋白质峰。与早中期相比,晚期肺癌患者血清中高表达的蛋白质相对分子质量为7978、8139、15 951和16 133,晚期肺癌患者血清中低表达的蛋白质相对分子质量为2867、6885、8701、8840、13 781、13 955。结论　SELDI-TOF-MS蛋白质芯片技术是一种快速、简单、敏感、样本用量少和高通量的蛋白质检查分析方法,可以直接筛选早中期和晚期肺癌患者血清中的差异蛋白,可能成为远处转移的诊断指标,具有较好的临床应用价值。     

Pentafluorobenzylation of alkyl and related DNA base adducts facilitates their determination by electrophore detection

The DNA adduct O4-ethylthymine can be alkylated under mild conditions with pentafluorobenzyl bromide. The product has good gas chromatographic characteristics and also forms a structurally characteristic anion in high yield when subjected to electron capture mass spectrometry. Related adducts for other DNA bases behave similarly. These properties stimulated us to develop a general analytical method based on the derivatization reaction. Important for this method is an oxidation-elimination reaction that mildly releases a base from a nucleoside. Thus, a general analytical method based on pentafluorobenzylation is now available for determining many alkyl and related DNA adducts.     

Synthesis, characterization and mutagenicity of 2-nitroso-6,7-dimethoxytetrahydroisoquinoline

The compound 6,7-dimethoxytetrahydroisoquinoline (Scheme 2, (ii] reacts with nitrous acid at ambient temperature and pH approximately 3.0 to give, in high yield the expected 2-nitroso-6,7-dimethoxytetrahydroisoquinoline (Scheme 2, (iii)). An unequivocal chemical structure of this nitroso derivative was established by high resolution mass spectrometry and 1H NMR spectrometry. Unlike many N-nitroso compounds, (iii) crystallizes in a single rotational isomer which spontaneously forms, in dimethylsulfoxide (DMSO) solution, an equilibrium mixture of the two E/Z isomeric forms with a t1/2 approximately 53 h. The compound is mutagenic to Salmonella tester strains TA98, TA100, TA1538, TA1535, and TA1537 but only in the presence of induced rat liver microsomes. The highest mutagenic activity of approximately 10 net revertants/nM was obtained with TA100 at a dose of 10 micrograms/plate. The compound (ii) is a close structural analog to the tetrahydroisoquinolines formed by endogenous condensation/cyclization reactions that can occur, between acetaldehyde and dopamine or norepinephrine, when alcohol is consumed.     

Stem cells and proteomics

With the accomplishment of the draft of human genome project and the sequencing of several decades of organisms and the adventure of post-genomics, proteomics, an important subject of post-genomics, has been applied in many fields such as biology, oncology and medicine, etc. People’s attention has gradually transferred from revealing the genetic information to discovering the functions of the genetic materials. In consideration of the limited number of genes and the relative stability of thei…     

Detection of adducts arising from human exposure to N-nitroso compounds

Humans are exposed to carcinogenic nitroso compounds (NOC), which are likely to result in the formation of DNA adducts. DNA adducts can be detected in human samples using a range of different analytical methodologies, including high pressure liquid chromatography (HPLC)-fluorescence, immunoassays and gas chromatography-mass spectrometry. Immunohistochemical studies offer the possibility of detecting adducts in single cells, but require further development for human studies. Sensitive 32P-postlabelling methods, in conjunction with HPLC separation, allow the detection of NOC-derived alkylated nucleotides in small samples of DNA derived from human tissues such as lymphocytes and placenta. In many studies, adducts have been detected in human DNA, but are often present, to a similar extent, in control and exposed subjects. In a number of studies, exposure to NOC has been inferred from the presence of characteristic alkyl adducts. In subjects from high risk areas for oesophageal cancer, DNA from target tissue contained higher levels of 0(6)-methyldeoxyguanosine than controls. The analysis of adducts in 'surrogate' DNA from peripheral lymphocytes appears promising as an accessible measure of alkylation damage. Also, the measurement of excreted levels of alkylpurines has the potential to be a noninvasive indication of short-term exposure to NOC. Endogenous synthesis of NOC can occur by a number of possible pathways in humans, and measurements of adducts will be a means of detecting the resulting alkylating agents, since their direct detection would be extremely difficult.     

Application of metabolomics in clinical and laboratory gastrointestinal oncology

Metabolites are versatile bioactive molecules. They are not only the substrates and/or the products of enzymatic reactions but also act as the regulators in the systemic metabolism. Metabolomics is a high-throughput analytical strategy to qualify or quantify as many metabolites as possible in the metabolomes. It is an indispensable part of systems biology. The leading techniques in this field are mainly based on mass spectrometry and nuclear magnetic resonance spectroscopy. The metabolomic analysis has gained wide use in bioscience fields. In the tumor research arena, metabolomics can be employed to identify biomarkers for prediction, diagnosis, and prognosis. Chemotherapeutic effect evaluation and personalized medicine decision-making can also benefit from metabolomic analysis of patient biofluid or biopsy samples. Many cell-level studies can help in disease exploration. In this review, the basic features and principles of varied metabolomic analysis are introduced. The value of metabolomics in clinical and laboratory gastrointestinal cancer studies is discussed, especially for mass spectrometry applications. Besides, combined use of metabolomics and other tools to solve problems in cancer practice is briefly illustrated. In summary, metabolomics paves a new way to explore cancerous diseases in the light of small molecules.     

基于质谱的尿蛋白质组学在肿瘤生物标志物发现中的研究进展

基于质谱（mass spectrometry,MS）的蛋白质组学技术的快速发展,应用蛋白质组学方法寻找新的肿瘤生物标志物成为肿瘤研究的热点。尿液以各种形式收集血液在稳态机制控制下排出的废物,其积累了人体生物系统的变化,能够很好地反映机体疾病的状态。相较于目前临床检测常用的血液样本,尿液样本具有成分简单稳定、可连续收集、更易检测感兴趣的低丰度蛋白等诸多优势,这使其成为肿瘤生物标志物研究的理想来源。尿液蛋白大多为分泌蛋白,因此在尿液中发现的潜在蛋白也可能是血液中很好的生物标志物。本文总结了基于MS的尿蛋白质组学在肿瘤生物标志物发现中的研究进展,并对常见肿瘤中筛选的尿蛋白标志物候选分子进行了系统归纳和分析。      

蛋白质组学在结直肠癌生物标志物研究中的应用

蛋白质组学是以蛋白质组为研究对象的一门崭新的生命科学,其技术主要包括样品的制备技术、双向凝胶电泳、差异凝胶电泳、多维蛋白质鉴定技术和表面增强激光解析电离飞行时间质谱等。结直肠癌是人类最常见的恶性肿瘤之一,近几年蛋白质组学在结直肠癌生物标志物的研究中取得了很大进展。本文对此作一综述。     

安罗替尼在晚期恶性肿瘤治疗中的研究进展

安罗替尼是我国自主研发的多靶点小分子酪氨酸激酶抑制剂,具有抗肿瘤血管生成和抑制肿瘤细胞生长的作用,因其具有不良反应小、靶点明确及安全性高等优点,目前已在多种恶性肿瘤中显示出了良好的临床疗效,本文就安罗替尼的抗肿瘤作用机制、其在多种类型肿瘤中的应用以及不良反应与管理等方面的最新研究进展进行综合阐述。     

Is it possible to make a diagnosis of cancer by smelling?

It had been reported that a house dog could make notify the dog's owner to have malignant melanoma through sniffing intently and trying to bite off a pigment lesion on the thigh of its owner. This fact resulted in extensive study of training dogs to distinguish the special odour of exhaled breath or urine of patients with cancer from those of the healthy subjects. These studies have promoted further development of detecting and identifying the special odour substances excreted from the patients with cancer using Gas chromatography-Mass spectrometry (GC-MS) technique and further expanded to the exploitation of the electronic noses. Here, the authors made a brief general survey on these progressions. Though the field of electronic noses has made a great progress in recent years, there remain many difficulties in predicting cancer with excellent accuracy. We hope that there should be a breakthrough in this field and every clinic could obtain a useful instrument at a moderate price and, in the near future, could predict with high reliability whether a patient has cancer or not.