质谱技术及其在临床检验中的应用
韩丽乔, 庄俊华, 黄宪章. 质谱技术及其在临床检验中的应用. ): 252-256&&
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质谱技术及其在临床检验中的应用
广州中医药大学第二临床医学院检验科,广东 广州 510120
作者简介:韩丽乔,女,1986年生,学士,主要从事临床生物化学研究。通讯作者:庄俊华,联系电话:020-08。
中图分类号:O657.62
文献标志码:A
文章编号:13)03-252-05
引言质谱(mass spectrometry,MS)技术是一种重要的检测分析技术,通过将待测样本转换成高速运动的离子,根据不同的离子拥有不同的质荷比(m/z)进行分离和检测目标离子或片段,然后依据保留时间和其丰度值进行定性和定量[]。近年来,质谱技术发展迅速,通过改进离子源和分离器相继发展了多种类型的质谱仪如电喷雾离子源质谱(ESI-MS)、大气压化学电离离子源质谱(APCI-MS)、四级杆(QQQ)质谱仪、离子阱质谱技术以及各种串联、联用质谱仪等多种类型,极大提高了检测的分辨率和检测范围。质谱技术最先应用于计量和分析化学领域,在临床检验中质谱仍属于一种年轻的检测方法。但自从其在临床检验应用以来,便以其高灵敏度、低检测限、样本用量少、高通量、检测速度快、样本前处理简单的优势显示出巨大的生命力,尤其和气相、高效液相色谱仪的联用极大的扩展了质谱技术在临床检验中的分析范围。一、质谱仪的组成质谱仪主要有5个部分组成:进样系统、离子源、质量分析器、检测器和数据处理系统。其核心部件是离子源和质量分析器。离子源的功能是将由进样系统引入的样本分子转化成离子,包括硬电离方法和软电离方法。硬电离方法给予样本较大的能量,如电子轰击电离、化学电离、场致离子化电离等;软电离方法是一种比较温和的离子化方式,包括快原子轰击电离、大气压化学电离、大气压光致电离、电喷雾电离、基质辅助激光解吸电离等类型。硬电离方法适用于一些小分子化合物的分析,软电离适用于分子量较大的化合物,尤其是一些生物分子,如蛋白质、多肽、寡聚核苷酸等。质量分析器主要是将电离产生的离子根据其不同的质荷比来分离目标离子,其主要类型有单聚焦、双聚焦、摆线、磁分析器、飞行时间、四级杆质量分析器、离子阱分析器、傅立叶变换离子回旋共振质谱等类型。此外仪器还需要在高真空环境中进行离子分离,因此真空系统也是质谱仪必备的组成部分[]。二、质谱仪的工作原理质谱仪的基本工作原理:待测样本由进样系统进入离子源内电离成离子进入质量分析器,然后质量分析器据形成的离子的m/z进行分离,后进入检测器检测,数据系统将离子信号转换成谱图进行质谱解析或定量分析。目前生命科学领域中的质谱仪大都由几种质量检测器串联组成,这样可以提高离子分离效率,使检测更具特异性和准确度[]。三、质谱仪在临床检验中应用(一)在微生物检验方面的应用传统的致病微生物检测大多采用微生物培养、生物化学和分子生物学的方法检测,不仅分析周期长而且没有明确的种群分型标准,往往造成分析结果的滞后和种类分型的误判。据估计,在临床实验室中仅在链球菌的分类中,就有高达13%的辨别错误[]。近年来,质谱技术在微生物检验方面的应用越来越多,这主要得益于其得天独厚的优势:(1)可用于多种微生物样本,如痰液、血液、尿液、脑脊液和胸腹腔积液以及经过培养的样本;(2)可用于几乎所有类型的病原体鉴定和分类检测,如细菌、真菌及其孢子、病毒、寄生虫等;(3)可对病原的多种成分进行分析,包括蛋白质、脂质、脂多糖、脂寡糖、DNA、多肽及其他可被离子化的分子;(4)检测速度快,例如一个病原微生物的质谱检定实验,包括样本的采集和制备,整个过程不到10 min[];(5)样本用量少;(6)样本前处理简单;(7)特异性和准确性高,例如金黄色葡萄球菌的表型鉴定,Rajakaruna等[]利用基质辅助激光解吸/电离-飞行时间质谱(SELDI-TOF-MS)技术分析了来自临床实验室的95个分离群和39个葡萄球菌群,并利用MicrobeLynx软件成功的识别了各个种群;(8)高敏感性,例如液相串联质谱可以检测到10~100个细菌或20~50个孢子的存在。在对生物样本进行处理后,甚至可以在单个菌水平发现并确定致病菌[] ,使其在微生物尤其是传染病病原体鉴定方面具有巨大的优势。近年来已建立了微生物胞膜蛋白质、脂多糖、核酸等的指纹数据库,使其检测更加准确和快速。目前在细菌检测中应用较多是飞行质谱技术。通过检测细菌胞膜成分或表达的特异蛋白对细菌进行种群的鉴别,不仅可以识别病原菌,而且有助于发现新的病原菌。此外还有用于病原体的药物敏感性实验检测和真菌检测研究等[,] 。(二)质谱技术在临床免疫学检验的应用飞行质谱技术的全称是表面增强激光解吸电离飞行时间质谱技术(surface enhanced laser desorption/ionization time of flight mass spectrometry,SELDI-TOF-MS),是利用相同能量的带电粒子,由于质量的差异而具有不同速度从而以不同时间通过相同的漂移距离到达接收器,依据离子束到达检测器的时间推算出m/z,从而进行定性和定量检测。其高灵敏度、高通量的分析特点使其在临床免疫学检验生物标志物检测方面成为一项有力的工具,筛选作用独特高效。如前列腺癌及前列腺增生、卵巢癌、胰腺癌、膀胱癌、乳腺癌、肺癌、肝癌、肾癌、结肠癌、喉癌、鼻咽癌、食道癌等,都发现了特异的蛋白或某些蛋白的增加或者减少[,,,]。尤其质谱检测技术在泌尿系统中的应用发展迅速。由于其检测的样本为尿液,对患者身体不造成伤害,不仅可以早期诊断肾脏疾病,而且避免或减轻了传统侵入性方法如肾脏活检等给患者带来的痛苦,提高患者治疗的依从性和存活率。在肾脏肿瘤、糖尿病性肾脏病变、肾脏移植功能检测等具有很大应用前景[]。此外毛细管电泳质谱技术被广泛用于泌尿系统 peptidomical 生物标志物的研究,验证了许多已经报道的但是没有蛋白和肽段识别的研究结果[]。Agger等[]采用液相色谱-多重反应检测-质谱(LC-MRM/MS)方法通过同时定量测定血浆样本中载脂蛋白A-Ⅰ和载脂蛋白B来推进质谱方法同时定量多种血浆/血清中蛋白质在临床前期的生物标志物大规模筛选以及取代价格昂贵的免疫测定方法作为实验室检查的常规方法的应用。结果表明LC-MRM/MS与传统免疫学方法相关性良好且具有大规模应用于临床研究的可行性。因其简化了样本的前处理以及样本前处理中蛋白消化作用带来的变异,具有潜在的应用前景。(三)在临床生物化学检验中的应用1.在体内激素检测方面的应用 质谱技术在临床生物化学中一项重要的应用是用于体内激素的检测,如类固醇激素(甾体激素)及其代谢产物的检测,具有极重要的临床诊断价值,几乎可以诊断所有的类固醇相关障碍性疾病。如睾酮(T)、双氢睾酮(DHT)、血浆雌酮硫酸盐、雌酮、雌二醇和雌三醇等的定量检测,可辅助多种激素相关疾病及激素替代治疗疾病如儿科遗传性激素相关疾病、先天性肾上腺增生症、家族性高醛甾酮过多症、多囊卵巢病、成人生殖系统和第二性征的维持、前列腺增生和前列腺癌、原发性醛固酮增多症、肾上腺机能减退、雌激素缺乏及抗雌激素药治疗、肾上腺功能异常(如Cushing's综合症)的诊断、监测、治疗和研究等[,]。2.在血药浓度监测和药物代谢研究中的应用 临床中某些药物效用范围比较窄,很容易引起毒性反应,造成不良后果。如免疫抑制剂随着器官移植技术和移植成活率的提高越来越多的应用于患者,其在人体内过多和过少都会给患者带来很大的痛苦。但这些药物在体内的浓度往往很低,给检测带来一定的困难。近几年随着质谱技术的发展,其高灵敏度、高特异性和检测速度快的优势使其已成为药物浓度检测的重要工具。特别是在免疫抑制药物、抗肿瘤药物、抗逆转录病毒(HIV)药物、抗精神病药物、一些激素类药物、中药及其天然产物分析、药物滥用(如吗啡、鸦片和一些镇痛药)、麻醉药、中毒药物的急救中得到广泛应用,并且质谱技术被公认为生物样本中药物及其代谢产物检测的标准化方法[,]。此外有报道通过用飞行质谱方法进行单链核苷酸多态性的快速基因分型从而指导华法林(抗凝血药)用量的研究[]。这为新的多重基因分型方法提供了一个非常好的临床检验平台,促进个体化药物治疗的研究。3.在遗传性疾病检测中的应用 质谱技术在遗传性疾病的诊断和筛查中应用广泛。最为大家熟知的就是质谱技术在新生儿筛查检测中的应用,通过检测氨基酸、脂肪酸、有机酸及其代谢产物可以灵敏、准确地检测出20多种遗传代谢疾病,从而早期诊断、早期治疗,挽救了很多患儿的生命和人生[,]。还有报道质谱技术用于快速筛查嘌呤和嘧啶代谢紊乱高危患者的研究,快速且特异,弥补了该种遗传病表型表现多样性和非特异性给诊断带来的困难[]。孙卫华等[]采用气相色谱质谱联用技术(GC-MS)测定尿琥珀酰丙酮(SA),精确性和准确性均较高,为临床上鉴别诊断酪氨酸血症I型提供了新的方法。4.痕量元素/微量营养素检测中的应用 电感耦合等离子体质谱分析技术(ICP-MS)是20世纪80年代发展起来的无机元素分析检测技术,近年来在痕量、超痕量成分及同位素分析检测中广泛应用。ICP-MS是在样本处理前加入待测元素的同位素,利用其测定前后的丰度比例改变而达到测定目的。可以同时测定多种痕量元素,具有检测限低、动态线性范围宽、干扰少、稳定性好、分析精密度高、速度快、样本前处理简单、高通量等诸多优点,是最准确的无机元素分析方法之一。目前已广泛应用于血清、全血、尿液以及头发中铅(Pb)、砷(As)、铁(Fe)、硒(Se)、锌(Zn)等有害重金属元素和人体微量元素的测定,可辅助临床疾病和职业病的诊断和鉴别诊断等。但是其一个最大的缺点是昂贵的耗费以及某些重金属元素比如铬和铁等有较多的干扰[]。此外,质谱技术还可应用于人体微量营养元素的检测,如B12、维生素D等,对于诊断人体相关微量营养素异常导致的疾病具有重要临床应用意义。5.糖化血红蛋白的检测应用 糖化血红蛋白(HbA1c)被认为是诊断糖尿病的最好指标。Nakanishi等[]报道用电喷雾电离质谱法(ESI-MS)检测HbA1c并与传统方法比较,结果显示ESI-MS方法与传统方法的相关性高于96%,而且重复性非常好,在临床检验常规方法的质量控制中可以起到很大作用。(四)在分子生物诊断中的应用1.蛋白组学和核苷酸多态性的研究应用 质谱技术应用领域中的另一个重要方面是蛋白质组学研究。2002年软电离技术被授予诺贝尔化学奖。生物质谱技术也成为质谱学中最具有活力的前沿热门技术。其可以检测蛋白质的氨基酸组成、分子量、多肽或二硫键的数目和位置及蛋白质的空间构象等;还用于检测核酸的分子量和单核苷酸多态性(即基因位点的突变)研究。其准确、灵敏和高通量的特点已经成为检测蛋白及多肽分子和基因的重要技术[,]。通过MALDI-TOF-MS检测寻找特异的一组蛋白质峰,建立肿瘤早期血清差异表达蛋白的诊断模型,对早期快速诊断肿瘤提供可能。已有研究报道用于多种肿瘤的早期诊断。单核苷酸多态性是指DNA序列上发生的单个核苷酸碱基之间的变异,在人群中的发生频率>1%,是决定疾病易感性和药物反应性差异的重要因素。通过检测突变的位点可以对疾病进行预测,提供诊断意见和用药指导并探讨与疾病发生的相关性。因为MALDI-TOF-MS检测的是核苷酸本身的分子量,相较于传统的单核苷酸多态性分型检测方法更为省时、省力和可靠。2.代谢组学研究的应用 人体是一个复杂的生化大工厂。当某部件出现异常时必然会伴随着某些代谢小分子的水平异常。相对来说这些小分子比DNA、RNA、蛋白质等更能反应一个细胞当前的功能状态。代谢组学也是目前研究的一个热点,质谱技术在检测这些代谢小分子的变化上也有重要的应用,为多种疾病及肿瘤的更早期诊断和指导治疗提供依据[]。如张文亮等[]采用毛细管电泳与MALDI-TOF-MS联用测定血清转甲状腺素蛋白的化学修饰,为进一步探索淀粉样变性的发病机制提供了一种简便的检测方法。(五)在参考方法建立和研制标准物质方面的应用在临床检验中基于准确性的标准化检测是目前急需的。1997年国际物质量咨询委员会(CCQM)将同位素稀释质谱(ED-ID-MS)原理定为一级(基准)测量原理之一,其同时具有质谱分析的高度特异性和同位素稀释的高度精密性,且测量的动态范围宽,样本制备不需严格定量操作,测量值能够直接溯源到国际单位制的物质量基本单位“摩尔”。因此基于同位素稀释质谱原理的方法在生物和临床化学溯源研究中受到越来越多的重视,为临床检验中标准物质的研制提供了技术保障,是临床检验参考方法的最佳选择。Thienpont 等[]建立基于常规实验室的ED-ID-MS法检测游离T3和T4通过IFCC的候选国际传统参考测量程序方法的校准和溯源,检测指标都取得了很好的相关性。张传宝等[]应用同位素稀释液相色谱串联质谱法建立了测定血清尿酸的候选参考方法。四、结语质谱技术,尤其是串联质谱技术可以提供物质的结构和质量信息。因此其在定性和定量生物样本中的作用越来越大,同时也适合一些探索性的工作。MALDI-TOF和SELDI质谱仪近来被广泛用于一些标志性物质的探索。三重四级杆线性离子阱分析器的发展使分析器定量的能力与离子阱的扫描能力相结合,从而可以容纳扫描器组合的不同排列。选择性反应物探测的组合和三重质谱技术的组合(MS/MS/MS)是一个定量的很好组合。电喷雾离子化、大气压光致电离是近年来发展迅速的离子源,可以检测极性和非极性化合物。质谱技术虽然有很多的优点,在近年来很多领域的应用也发展迅速,但其也有自身的瓶颈:如没有某纯物质为内标或特征性的离子碎片,则难以判断该物质是何种物质,无法定性和定量,所以目前还有许多物质无法用质谱检测,尤其是一些大分子的复杂物质;目前质谱技术的自动化程度还还相对较差,前处理过程也相对复杂,其对工作人员的技术要求较高;另外仪器昂贵,日常运行费用及维护费用也较高,如ID-MS仪器,在处理样本时需要加入适量的同位素稀释剂,该种稀释剂来源较困难,制备成本较高等,这些都为ID-MS的普及应用带来困难;此外该技术的高敏感性,如SELDI-TOF-MS技术筛检蛋白的高敏感性必然带来了检测的假阳性,这也是该技术不容忽视的一个弱点。但相信随着质谱技术的发展成熟,其在临床实验室检测中会有更广泛的应用。
The authors have declared that no competing interests exist.
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[本文引用:1]
... 引言质谱(mass spectrometry,MS)技术是一种重要的检测分析技术,通过将待测样本转换成高速运动的离子,根据不同的离子拥有不同的质荷比(m/z)进行分离和检测目标离子或片段,然后依据保留时间和其丰度值进行定性和定量[1] ...
... 此外仪器还需要在高真空环境中进行离子分离,因此真空系统也是质谱仪必备的组成部分[2] ...
... 目前生命科学领域中的质谱仪大都由几种质量检测器串联组成,这样可以提高离子分离效率,使检测更具特异性和准确度[3] ...
... 据估计,在临床实验室中仅在链球菌的分类中,就有高达13%的辨别错误[4] ...
... (4)检测速度快,例如一个病原微生物的质谱检定实验,包括样本的采集和制备,整个过程不到10 min[5] ...
. ):507-513
Staphylococcus aureus remains an important human pathogen responsible for a high burden of disease in healthcare and community settings. The emergence of multidrug-resistant strains is of increasing concern world-wide. The identification of S. aureus is currently based upon phenotypic and genotypic methods. Here, an alternative approach involving mass spectral analysis of surface-associated proteins of intact bacterial cells by matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF-MS) was investigated using 95 isolates obtained directly from a clinical laboratory at The Royal London Hospital and 39 isolates from the Staphylococcal Reference Unit, Health Protection Agency, London. Results obtained indicate that clinical isolates share many common mass ions with-type/reference strains which allowed their correct identification when searched against a comprehensive database that has been in the process of development for several years. The existing database contains more than 5000 profiles of various bacterial pathogens, but comprises mainly type or reference strains. The MicrobeLynx software successfully identified all isolates to the correct genus and all but four to the correct species. These were misidentified in the first instance due to contamination or low mass ion intensity but once the cultures were purified and re-analysed they were confirmed as S. aureus by both MALDI-TOF-MS and 16S rRNA sequence analysis. The high percentage of correct identifications coupled with the high speed and the minimal sample preparation required, indicate that MALDI-TOF-MS has the potential to perform high throughput identification of clinical isolates of S. aureus despite the inherent diversity of this species. The method is, however, only reproducible if variable parameters such as sample preparation, media, growth condition, etc. are standardised.
... (7)特异性和准确性高,例如金黄色葡萄球菌的表型鉴定,Rajakaruna等[6]利用基质辅助激光解吸/电离-飞行时间质谱(SELDI-TOF-MS)技术分析了来自临床实验室的95个分离群和39个葡萄球菌群,并利用MicrobeLynx软件成功的识别了各个种群 ...
... 在对生物样本进行处理后,甚至可以在单个菌水平发现并确定致病菌[7] ,使其在微生物尤其是传染病病原体鉴定方面具有巨大的优势 ...
PLoS ONE. ):e25712-null
Sebastian van Hal 2 ,Wieland Meyer 1,3 ,Sharon C.-A. Chen 1,3 ,Tom Olma 1 ,Angie Pinto 1 ,Melissa Zahra 2 ,Krystyna Maszewska 3 ,Catriona Halliday 1 ,Jonathan R. Iredell 1
&sup&1&/sup&
Centre for Infectious Diseases and Microbiology Laboratory Services, Westmead Hospital, Sydney, New South Wales, Australia&br/&&sup&2&/sup&
Department of Microbiology and Infectious Diseases, Sydney South West Pathology Service, Liverpool, Sydney, New South Wales, Australia&br/&&sup&3&/sup&
Molecular Mycology Research Laboratory, Westmead Millenium Institute, Sydney Medical School – Westmead Hospital, University of Sydney, Sydney, New South Wales, Australia&br/&
BackgroundMatrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for yeast identification is limited by the requirement for protein extraction and for robust reference spectra across yeast species in databases. We evaluated its ability to identify a range of yeasts in comparison with phenotypic methods.MethodsMALDI-TOF MS was performed on 30 reference and 167 clinical isolates followed by prospective examination of 67 clinical strains in parallel with biochemical testing (total n = 264). Discordant/unreliable identifications were resolved by sequencing of the internal transcribed spacer region of the rRNA gene cluster.Principal FindingsTwenty (67%; 16 species), and 24 (80%) of 30 reference strains were identified to species, (spectral score ≥2.0) and genus (score ≥1.70)-level, respectively. Of clinical isolates, 140/167 (84%) strains were correctly identified with scores of ≥2.0 and 160/167 (96%) with scores of ≥1.70; amongst Candida spp. (n = 148), correct species assignment at scores of ≥2.0, and ≥1.70 was obtained for 86% and 96% isolates, respectively (vs. 76.4% by biochemical methods). Prospectively, species-level identification was achieved for 79% of isolates, whilst 91% and 94% of strains yielded scores of ≥1.90 and ≥1.70, respectively (100% isolates identified by biochemical methods). All test scores of 1.70–1.90 provided correct species assignment despite being identified to “genus-level”. MALDI-TOF MS identified uncommon Candida spp., differentiated Candida parapsilosis from C. orthopsilosis and C. metapsilosis and distinguished between C. glabrata, C. nivariensis and C. bracarensis. Yeasts with scores of <1.70 were rare species such as C. nivariensis (3/10 strains) and C. bracarensis (n = 1) but included 4/12 Cryptococcus neoformans. There were no misidentifications. Four novel species-specific spectra were obtained. Protein extraction was essential for reliable results.ConclusionsMALDI-TOF MS enabled rapid, reliable identification of clinically-important yeasts. The addition of spectra to databases and reduction in identification scores required for species-level identification may improve its utility.
PLoS ONE. ):e16833-null
Stefan Balabanov 1 ,Frederike Mundt 1 ,Tim H. Brümmendorf 1,10 ,Christine Barett 1 ,Maria F?lth 6 ,Heike Pospisil 2 ,Simone Venz 3,4 ,Jens K?llermann 7 ,Holger Sültmann 6 ,A. Schuppert 9 ,Carsten Bokemeyer 1 ,Thorsten Schlomm 8 ,Guido Sauter 7 ,Ramesh Ummanni 1 ,Reinhard Walther 3 ,Christian Scharf 4,5
&sup&1&/sup&
Department of Oncology, Haematology and Bone Marrow Transplantation with Section Pneumology, Hubertus Wald-Tumor Zentrum, University Hospital Eppendorf, Hamburg, Germany&br/&&sup&2&/sup&
Bioinformatics, University of Applied Sciences Wildau, Wildau, Germany&br/&&sup&3&/sup&
Department of Medical Biochemistry and Molecular Biology, University of Greifswald, Greifswald, Germany&br/&&sup&4&/sup&
Interfacultary Institute of Genetics and Functional Genomics, University of Greifswald, Greifswald, Germany&br/&&sup&5&/sup&
Department of Otorhinolaryngology, Head and Neck Surgery, University of Greifswald, Greifswald, Germany&br/&&sup&6&/sup&
Cancer Genome Research, Deutsches Krebsforschungszentrum, Heidelberg, Germany&br/&&sup&7&/sup&
Department of Pathology, University Hospital Eppendorf, Hamburg, Germany&br/&&sup&8&/sup&
Prostate Cancer Center, University Hospital Eppendorf, Hamburg, Germany&br/&&sup&9&/sup&
Aachen Institute for Advanced Study in Computational Engineering Science, RWTH Aachen University, Aachen, Germany&br/&&sup&10&/sup&
Medizinische Klinik IV - H?matologie und Onkologie, RWTH Aachen University, Aachen, Germany&br/&
Prostate cancer (PCa) is the most common type of cancer found in men and among the leading causes of cancer death in the western world. In the present study, we compared the individual protein expression patterns from histologically characterized PCa and the surrounding benign tissue obtained by manual micro dissection using highly sensitive two-dimensional differential gel electrophoresis (2D-DIGE) coupled with mass spectrometry. Proteomic data revealed 118 protein spots to be differentially expressed in cancer (n = 24) compared to benign (n = 21) prostate tissue. These spots were analysed by MALDI-TOF-MS/MS and 79 different proteins were identified. Using principal component analysis we could clearly separate tumor and normal tissue and two distinct tumor groups based on the protein expression pattern. By using a systems biology approach, we could map many of these proteins both into major pathways involved in PCa progression as well as into a group of potential diagnostic and/or prognostic markers. Due to complexity of the highly interconnected shortest pathway network, the functional sub networks revealed some of the potential candidate biomarker proteins for further validation. By using a systems biology approach, our study revealed novel proteins and molecular networks with altered expression in PCa. Further functional validation of individual proteins is ongoing and might provide new insights in PCa progression potentially leading to the design of novel diagnostic and therapeutic strategies.
. ):945-961
Ajay Matta 1 ,Ranju Ralhan 1,2,3,4,5 ,Leroi V. DeSouza 1 andK.W. Michael Siu 1,*
&span class="affiliationNumber"&1&/span&Department of Chemistry, Centre for Research in Mass Spectrometry, York University, 4700 Keele Street, Toronto, Ontario, Canada M3J 1P3&br/&&span class="affiliationNumber"&2&/span&Joseph and Mildred Sonshine Family Centre for Head and Neck Disease, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, Canada M5G 1X5&br/&&span class="affiliationNumber"&3&/span&Department of Otolaryngology-Head and Neck Surgery, Mount Sinai Hospital, Toronto, Ontario, Canada M5G 2N2&br/&&span class="affiliationNumber"&4&/span&Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada&br/&&span class="affiliationNumber"&5&/span&Department of Otolaryngology-Head and Neck Surgery, University of Toronto, Toronto, Ontario, Canada M5G 2N2&br/&&sup&*&/sup&Department of Chemistry and Centre for Research in Mass Spectrometry, York University, 4700 Keele Street, Toronto, Ontario, Canada M3J 1P3.
>Mass spectrometry (MS)-based proteomics is a rapidly developing technology for both qualitative and quantitative analyses of proteins, and investigations into protein posttranslational modifications, subcellular localization, and interactions. Recent advancements in MS have made tremendous impact on the throughput and comprehensiveness of cancer proteomics, paving the way to unraveling deregulated cellular pathway networks in human malignancies. In turn, this knowledge is rapidly being translated into the discovery of novel potential cancer markers (PCMs) and targets for molecular therapeutics. Head-and-neck cancer is one of the most morbid human malignancies with an overall poor prognosis and severely compromised quality of life. Early detection and novel therapeutic strategies are urgently needed for more effective disease management. The characterizations of protein profiles of head-and-neck cancers and non-malignant tissues, with unprecedented sensitivity and precision, are providing technology platforms for identification of novel PCMs and drug targets. Importantly, low-abundance proteins are being identified and characterized, not only from the tumor tissues, but also from bodily fluids (plasma, saliva, and urine) in a high-throughput and unbiased manner. This review is a critical appraisal of recent advances in MS-based proteomic technologies and platforms for facilitating the discovery of biomarkers and novel drug targets in head-and-neck cancer. A major challenge in the discovery and verification of these cancer biomarkers is the typically limited availability of well-characterized and adequately stored clinical samples in tumor and sera banks, collected using recommended procedures, and with detailed information on clinical, pathological parameters, and follow-up. Most biomarker discovery studies use limited number of clinical samples and verification of cancer markers in large number of samples is beyond the scope of a single laboratory. The validation of these potential markers in large sample cohorts in multicentric studies is needed for their translation from the bench to the bedside. & 2010 Wiley Periodicals, Inc. Mass Spec Rev 29:945&961, 2010
... 在肾脏肿瘤、糖尿病性肾脏病变、肾脏移植功能检测等具有很大应用前景[14] ...
. ):null-null
... 此外毛细管电泳质谱技术被广泛用于泌尿系统 peptidomical 生物标志物的研究,验证了许多已经报道的但是没有蛋白和肽段识别的研究结果[15] ...
... Agger等[16]采用液相色谱-多重反应检测-质谱(LC-MRM/MS)方法通过同时定量测定血浆样本中载脂蛋白A-#cod#x02160 ...
. -2):null-null
. ):162-168
Warfarin exhibits significant interindividual variability in dosing requirements. Different drug responses are partly attributed to the single nucleotide polymorphisms (SNPs) that influence either drug action or drug metabolism. Rapid genotyping of these SNPs helps clinicians to choose appropriate initial doses to quickly achieve anticoagulation effects and to prevent complications. We report a novel application of surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF MS) in the rapid genotyping of SNPs that impact warfarin efficacy. The SNPs were first amplified by PCR and then underwent single base extension to generate the specific SNP product. Next, genetic variants displaying different masses were bound to Q10 anionic proteinChips and then genotyped by using SELDI-TOF MS in a multiplex fashion. SELDI-TOF MS offered unique properties of on-chip sample enrichment and clean-ups, which streamlined the testing procedures and eliminated many tedious experimental steps required by the conventional MS-based method. The turn-around time for genotyping three known warfarin-related SNPs, CYP2C9*2, CYP2C9*3, and VKORC1 3673G>A by SELDI-TOF MS was less than 5 hours. The analytical accuracy of this method was confirmed both by bidirectional DNA sequencing and by comparing the genotype results (n = 189) obtained by SELDI-TOF MS to reports from a clinical reference laboratory. This new multiplex genotyping method provides an excellent clinical laboratory platform to promote personalized medicine in warfarin therapy.
... 此外有报道通过用飞行质谱方法进行单链核苷酸多态性的快速基因分型从而指导华法林(抗凝血药)用量的研究[21] ...
... 还有报道质谱技术用于快速筛查嘌呤和嘧啶代谢紊乱高危患者的研究,快速且特异,弥补了该种遗传病表型表现多样性和非特异性给诊断带来的困难[24] ...
... Nakanishi等[24]报道用电喷雾电离质谱法(ESI-MS)检测HbA1c并与传统方法比较,结果显示ESI-MS方法与传统方法的相关性高于96%,而且重复性非常好,在临床检验常规方法的质量控制中可以起到很大作用 ...
... 孙卫华等[25]采用气相色谱质谱联用技术(GC-MS)测定尿琥珀酰丙酮(SA),精确性和准确性均较高,为临床上鉴别诊断酪氨酸血症I型提供了新的方法 ...
. ):null-null
... 但是其一个最大的缺点是昂贵的耗费以及某些重金属元素比如铬和铁等有较多的干扰[26] ...
. ):null-null
Biochemistry. ):-null
Yuanqi Tao
Ryan R. Julian
&div id="AFF-d1455e40-autogenerated"&Department of Chemistry, &span class="institution"&University of California&/span&, Riverside, California 92521, United States&/div&&div&*Phone: &span class="phone"&(951) 827-3958&/span&. E-mail: &a href="mailto:ryan.julian@ucr.edu"&ryan.julian@ucr.edu&/a&.&/div&
A simple mass spectrometry-based method capable of examining protein structure called SNAPP (selective noncovalent adduct protein probing) is used to evaluate the structural consequences of point mutations in naturally occurring sequence variants from different species. SNAPP monitors changes in the attachment of noncovalent adducts to proteins as a function of structural state. Mutations that lead to perturbations to the electrostatic surface structure of a protein affect noncovalent attachment and are easily observed with SNAPP. Mutations that do not alter the tertiary structure or electrostatic surface structure yield similar results by SNAPP. For example, bovine, porcine, and human insulin all have very similar backbone structures and no basic or acidic residue mutations, and the SNAPP distributions for all three proteins are very similar. In contrast, four variants of cytochrome c (cytc) have varying degrees of sequence homology, which are reflected in the observed SNAPP distributions. Bovine and pigeon cytc have several basic or acidic residue substitutions relative to horse cytc, but the SNAPP distributions for all three proteins are similar. This suggests that these mutations do not significantly influence the protein surface structure. On the other hand, yeast cytc has the least sequence homology and exhibits a unique, though related, SNAPP distribution. Even greater differences are observed for lysozyme. Hen and human lysozyme have identical tertiary structures but significant variations in the locations of numerous basic and acidic residues. The SNAPP distributions are quite distinct for the two forms of lysozyme, suggesting significant differences in the surface structures. In summary, SNAPP experiments are relatively easy to perform, require minimal sample consumption, and provide a facile route for comparison of protein surface structure between highly homologous proteins.
Matrix-assisted laser desorption-ionization (MALDI) mass spectrometry has evolved as a powerful method for analyzing nucleic acids. Here we provide protocols for genotyping single-nucleotide polymorphisms (SNPs) by MALDI based on PCR and primer extension to generate allele-specific products. Furthermore, we present three different approaches for sample preparation of primer-extension products before MALDI analysis and discuss their potential areas of application. The first approach, the 'GOOD' assay, is a purification-free procedure that uses DNA-modification chemistry, including alkylation of phosphorothioate linkages in the extension primers. The other two approaches use either solid-phase extraction or microarray purification for the purification of primer-extension products. Depending on the reaction steps of the various approaches, the protocols take about 6&#x02013;8 hours.
&li&&span class="position"&1.&/span&&span class="affiliation"&Division of Gastroenterology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-Cho, Chu-o-ku, Kobe, Hyogo, 650-0017, Japan&/span&&/li&&li&&span class="position"&2.&/span&&span class="affiliation"&The Integrated Center for Mass Spectrometry, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-Cho, Chu-o-ku, Kobe, Hyogo, 650-0017, Japan&/span&&/li&&li&&span class="position"&3.&/span&&span class="affiliation"&Division of Metabolomics Research, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-Cho, Chu-o-ku, Kobe, Hyogo, 650-0017, Japan&/span&&/li&&li&&span class="position"&4.&/span&&span class="affiliation"&Division of Lipid Biochemistry, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-Cho, Chu-o-ku, Kobe, Hyogo, 650-0017, Japan&/span&&/li&
AbstractRecently, metabolome analysis has been increasingly applied to biomarker detection and disease diagnosis in medical studies. Metabolome analysis is a strategy for studying the characteristics and interactions of low molecular weight metabolites under a specific set of conditions and is performed using mass spectrometry and nuclear magnetic resonance spectroscopy. There is a strong possibility that changes in metabolite levels reflect the functional status of a cell because alterations in their levels occur downstream of DNA, RNA, and protein. Therefore, the metabolite profile of a cell is more likely to represent the current status of a cell than DNA, RNA, or protein. Thus, owing to the rapid development of mass spectrometry analytical techniques metabolome analysis is becoming an important experimental method in life sciences including the medical field. Here, we describe metabolome analysis using liquid chromatography–mass spectrometry, gas chromatography–mass spectrometry (GC–MS), capillary electrophoresis–mass spectrometry, and matrix assisted laser desorption ionization–mass spectrometry. Then, the findings of studies about GC–MS-based metabolome analysis of gastroenterological diseases are summarized, and our research results are also introduced. Finally, we discuss the realization of disease diagnosis by metabolome analysis. The development of metabolome analysis using mass spectrometry will aid the discovery of novel biomarkers, hopefully leading to the early detection of various diseases.
... 代谢组学也是目前研究的一个热点,质谱技术在检测这些代谢小分子的变化上也有重要的应用,为多种疾病及肿瘤的更早期诊断和指导治疗提供依据[30] ...
... 如张文亮等[31]采用毛细管电泳与MALDI-TOF-MS联用测定血清转甲状腺素蛋白的化学修饰,为进一步探索淀粉样变性的发病机制提供了一种简便的检测方法 ...
... Thienpont 等[32]建立基于常规实验室的ED-ID-MS法检测游离T3和T4通过IFCC的候选国际传统参考测量程序方法的校准和溯源,检测指标都取得了很好的相关性 ...
... 张传宝等[32]应用同位素稀释液相色谱串联质谱法建立了测定血清尿酸的候选参考方法 ...
质谱技术及其在临床检验中的应用
[韩丽乔, 庄俊华, 黄宪章]}