生物质谱技术的发展及在蛋白质结构研究中的应用

综述生物质谱技术的发展现状，包括电喷雾电离质谱、基质辅助激光解吸电离质谱、串联质谱、电喷雾解吸电离质谱、表面增强激光解吸电离-飞行时间质谱等，以及这些技术在蛋白质结构研究中的应用进展。生物质谱技术已成为蛋白质结构研究的重要工具。     

蛋白质芯片-飞行质谱技术及其在临床医学中的应用

蛋白质组学研究是最终揭示生命现象本质的关键，如今该学科已步入生命科学研究的最前沿。与之相伴随着的生物高新技术发展催生了一项新型生物芯片技术，即蛋白质芯片飞行质谱技术——表面加强激光解析电离飞行时间质谱(surface enhanced laser desorption／ionization—time of flight—mass spectra，SELDI-TOF—MS)。该技术将蛋白质芯片和质谱技术相结合，集样品分离、纯化、检测和数据分析为一体，具快速和高通量，成为目前蛋白质表达及蛋白质组学研究的有力工具。其可望为众多疾病的机理阐明及攻克提供理论依据和解决途径，在临床疾病诊断和防治方面发挥重要作用，显示了良好的发展前景和广泛的应用前景。本文对蛋白质芯片-飞行质谱技术的原理、特点及部分疾病的基础和临床研究等进行文献综述。     

生物质谱技术在蛋白质结构鉴定中的应用进展

综述了生物质谱技术在蛋白质结构鉴定中的应用新进展,讨论了生物质谱在蛋白质初步鉴定、翻译后修饰的鉴别、蛋白质-蛋白质间相互作用等研究方面的应用趋势,以及一些最新发展,指出了生物质谱技术现存的主要问题,探讨了可能的解决方案。     

应用蛋白质质谱技术检测肺癌血清生物标志物的实验研究

目的探讨蛋白质质谱分析方法筛选肺癌血清蛋白特异性生物标志物的可行性。方法采用表面增强激光解析离子化飞行时间质谱技术(SELDI)和弱阳离子交换(WCX2)蛋白质芯片检测17只健康兔和23只荷瘤兔的血清蛋白质谱,后者取血时影像学检查15只肺内已有肿瘤形成(荷瘤组),8只未见肿瘤形成的实验动物在随访过程中均见肿瘤形成(癌前组)。使用PBSII-C型蛋白质芯片阅读机读取数据,获得的结果采用Bio-marker wizard和Proteinchip 3.1软件进行分析。结果荷瘤组、癌前组和健康对照组比较,筛选出来的32个蛋白质峰中有5个峰(标志分子)表达差异有统计学意义(P<0.05),其中3个蛋白质表达上调,2个下调。结论蛋白质质谱分析方法可以从血清中检测出与肿瘤病程进展相关的特异性标志分子,有望成为肺癌早期诊断或预警的有效工具。     

生物标志物分类及其在临床医学中的应用

随着生命科学的发展,生物标志物在医学中的应用越来越广泛。生物标志物是一种能够判断疾病发生、发展和预后的指示物,根据特性可将其分为小分子生物标志物、大分子生物标志物、复合生物标志物和生物种群标志物。生物标志物可用于疾病的诊断和分类,监测疾病的发展和严重程度,检验临床治疗效果,预测个体发病的风险,并可用于高危人群筛查。生物标志物的选择需要详尽和严格的临床验证,并考虑其在特定临床情况下使用的可行性和易用性。随着对生物标志物的深入研究,其应用逐步从单一化向组合化发展。将生物标志物与其他检测手段联合应用,有助于更加早期、快速而准确地诊断疾病和判断病情,从而为临床治疗提供依据。     

SELDI蛋白质芯片在肿瘤标志物筛选中的应用进展

随着人类进入后基因组时代,蛋白质组学成为生命科学的一大研究热点.蛋白质芯片作为蛋白质组学研究中一项有前景的技术,被广泛应用于肿瘤的标志物筛选、早期诊断、分期及疗效、预后评价方面.本文就表面增强激光解吸电离(surface enhanced laser desorption /ionization, SELDI)蛋白质芯片的原理及近年来在肿瘤标志物筛选中的应用做一简要综述.     

心肌损伤生物标志物在毒理学实验中的应用进展

心肌损伤生物标志物的检测在心脏疾病临床诊断上被广泛应用,但是在新药的非临床安全评价方面,心肌损伤标志物的应用相对滞后。目前中国、美国、经济合作发展组织(OECD)的新药安全评价指导原则推荐的检测心脏功能的指标为谷草转氨酶(AST)、乳酸脱氢酶(LDH)和肌酸激酶(CK)。但是上述指标敏感性差(如AST)以及存在同工酶(如LDH和CK),所以不能特异灵敏地反映心脏的功能。目前国际上高度推崇采用敏感效应生物标志物来监测心脏的靶器官毒性。近年来肌红蛋白(Mb)、脑型钠尿肽(BNP)、心肌肌钙蛋白(cTn)对心肌损伤的检测展现了良好的应用价值,本文将对上述3项国内外研究最热点的心肌损伤生物标志物进行阐述。     

质谱法在中、美、英、日及欧洲药典中的应用进展

通过查阅近年来发达国家或地区药典中收载品种、通用检测方法等相关内容,分析和比较了各种质谱法在《中国药典》《美国药典》《英国药典》《日本药局方》和《欧洲药典》中的应用及变化情况,拟为质谱法在我国法定质量控制体系中的深入应用提供思路。     

Mass Spectrometry Innovations in Drug Discovery and Development

This review highlights the many roles mass spectrometry plays in the discovery and development of new therapeutics by both the pharmaceutical and the biotechnology industries. Innovations in mass spectrometer source design, improvements to mass accuracy, and implementation of computer-controlled automation have accelerated the purification and characterization of compounds derived from combinatorial libraries, as well as the throughput of pharmacokinetics studies. The use of accelerator mass spectrometry, chemical reaction interface-mass spectrometry and continuous flow-isotope ratio mass spectrometry are promising alternatives for conducting mass balance studies in man. To meet the technical challenges of proteomics, discovery groups in biotechnology companies have led the way to development of instruments with greater sensitivity and mass accuracy (e.g., MALDI-TOF, ESI-Q-TOF, Ion Trap), the miniaturization of separation techniques and ion sources (e.g., capillary HPLC and nanospray), and the utilization of bioinformatics. Affinity-based methods coupled to mass spectrometry are allowing rapid and selective identification of both synthetic and biological molecules. With decreasing instrument cost and size and increasing reliability, mass spectrometers are penetrating both the manufacturing and the quality control arenas. The next generation of technologies to simplify the investigation of the complex fate of novel pharmaceutical entities in vitro and in vivo will be chip-based approaches coupled with mass spectrometry.     

Effects of Excipients on Protein Conformation in Lyophilized Solids by Hydrogen/Deuterium Exchange Mass Spectrometry

Purpose Excipients are added to lyophilized protein drug formulations to protect the protein during processing and storage, but the mechanisms are poorly understood. Here, hydrogen/deuterium (H/D) exchange with mass spectrometry was used to assess protein conformation and excipient interactions in lyophilized solids. Methods Calmodulin (CaM, 17 kD) was co-lyophilized with carbohydrate excipients (sucrose, mannitol, trehalose, raffinose, dextran 5,000, dextran 12,000) or guanidine hydrochloride (negative control) and exposed to D₂O vapor at 33% RH and RT. Samples were then dissolved and analyzed by mass spectrometry (+ESI/MS). Peptic digestion provided additional, site-specific information on H/D exchange. Solids were further characterized by powder x-ray diffraction (PXRD), differential scanning calorimetry (DSC), infrared spectroscopy (FTIR) and water vapor sorption. Results Excipients protected CaM from H/D exchange, increasing in the order guanidine hydrochloride < no excipient, mannitol < dextran 5,000, dextran 12,000 < sucrose < raffinose < trehalose. Effects were exerted primarily in the protein’s α-helical segments. Conclusions The effects of carbohydrate excipients on protein conformation in lyophilized solids are not exhibited uniformly along the protein sequence, but instead are exerted in a site-specific manner. The results also demonstrate the utility of H/D exchange with ESI/MS for protein structure characterization in lyophilized samples.     

Mass Spectrometry Imaging,an Emerging Technology in Neuropsychopharmacology

Mass spectrometry imaging is a powerful tool for directly determining the distribution of proteins, peptides, lipids, neurotransmitters, metabolites and drugs in neural tissue sections in situ. Molecule-specific imaging can be achieved using various ionization techniques that are suited to different applications but which all yield data with high mass accuracies and spatial resolutions. The ability to simultaneously obtain images showing the distributions of chemical species ranging from metal ions to macromolecules makes it possible to explore the chemical organization of a sample and to correlate the results obtained with specific anatomical features. The imaging of biomolecules has provided new insights into multiple neurological diseases, including Parkinson''s and Alzheimer''s disease. Mass spectrometry imaging can also be used in conjunction with other imaging techniques in order to identify correlations between changes in the distribution of important chemical species and other changes in the properties of the tissue. Here we review the applications of mass spectrometry imaging in neuroscience research and discuss its potential. The results presented demonstrate that mass spectrometry imaging is a useful experimental method with diverse applications in neuroscience.     

现代生物质谱技术在生物大分子分析研究中的应用

随着当今生物质谱技术的蓬勃发展,生物质谱已经成为分析,鉴定蛋白质、多肽、细胞因子等生物大分子的重要手段.笔者对基质辅助激光解吸离子化质谱(MALDI-MS),电喷雾离子化质谱(ESI-MS),飞行时间质谱(TOF-MS)和离子阱质谱(Ion trap-MS)等生物质谱的功能和应用以及现代生物质谱与液相(LC)、毛细管电泳(CE)和二维液相色谱(2D-LC)等联用技术的最新应用与进展作了简要的综述.     

质谱成像技术在中药代谢领域的应用

质谱成像技术是一种全面、快速的新型分析技术,在中药代谢领域具有其独特优势,可以解决由于中药化学成分多样,代谢产物繁杂难以分析,限制其代谢研究的这一科学问题。本文通过检索国内外相关文献,对质谱成像技术的分类、相关设备及发展进行介绍,重点阐述了不同原理的质谱成像技术在中药代谢领域的应用,同时对该技术现有的优势与发展进行了总结。     

基于气相色谱质谱联用技术的代谢组学在小儿原发性肾病综合征中的应用

目的:通过分析原发性肾病综合征(PNS)患儿血浆代谢物,构建与PNS相关的代谢途径网络,发现潜在的生物标志物以辅助临床诊断。方法:采用气相色谱质谱联用(GC-MS)技术分析22例PNS患儿及22例健康儿童血浆代谢物,利用预处理软件XCMS进行数据预处理,主成分分析(PCA)与偏最小二乘判别分析(PLS-DA)对两组差异性代谢产物进行分析。结果:构建了PCA与PLS-LDA模型,PCA模型中变量的可解释率和可预测率为43.6% 和12.9%,PLS鄄LDA 模型中变量的可解释率和可预测率分别为79.8%和59.2%,共筛选出15种差异性代谢物,主要有胆固醇、柠檬酸、亚油酸及氨基酸等,其中乳酸盐、亚油酸、软 脂酸、丙酮酸、胆固醇含量升高,其余10种代谢物含量降低。结论:筛选出的15种差异性代谢物变化导致糖酵解、糖异生、脂类代谢和蛋白质代谢减弱,三羧酸循环紊乱。该研究为进一步明确PNS发病机制提供了新的科学依据,并有助于疾病诊断和预后评估。     

Structural Characterization of a Recombinant Monoclonal Antibody by Electrospray Time-of-Flight Mass Spectrometry

Purpose The aim of this study was to perform structural characterization of a recombinant monoclonal antibody (MAb), huN901, by electrospray time-of-flight mass spectrometry (ESI-TOFMS) using both “top-down” and “bottom-up” approaches.Methods In the top-down approach, the molecular masses of the deglycosylated huN901 and the light and heavy chains of the antibody were measured by direct infusion MS and liquid chromatography–mass spectrometry (LC–MS). In the bottom-up approach, trypsin and Asp-N protease were used to digest the separated, reduced and alkylated light and heavy chains followed by LC–MS analysis of the digests.Results The primary structure and post-translational modifications of huN901 were characterized by both top-down and bottom-up MS approaches. Modifications of N-terminal pyroglutamate formation, cleavage of C-terminal lysine, glycosylation, and deamidation were identified in the antibody heavy chain by both protein mass measurement and peptide mapping. No modifications were found in the complementarity determining regions (CDRs) of both chains. Both trypsin and Asp-N protease digestion had an average sequence recovery of 97%, and generated complimentary mapping results with complete sequence recovery.Conclusions ESI-TOFMS is a superior tool to characterize MAb and other complex protein pharmaceuticals.