Mass Spectrometry Of Drugs, Natural Products, Proteins,

药物、天然产物、蛋白质的质谱分析，

• 批准号: 6673454
• 负责人: Lewis K. Pannell
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6673454
• 关键词: aminoacid; antiviral agents; biological products; chemical information system; crosslink; decarboxylases; disulfide bond; drug screening /evaluation; electrospray ionization mass spectrometry; glycosylation; human immunodeficiency virus; marine biology; mass spectrometry; method development; oligonucleotides; peptides; phosphorylation; posttranslational modifications; protein structure; sickle cell anemia; stereoisomer; structural biology

The Structural Mass Spectrometry Group now concentrates almost entirely on peptide and protein type samples. These may come to the laboratory as spots on gels, recombinant protein samples for structural confirmation or post-translational modification determination, as proteins or protein complexes precipitated or purified with antibodies, or as entire proteomes. Spots on gels are routinely identified by digestion of the spot with trypsin, after reduction and alkylation, and subsequent analysis on the QTOF mass spectrometer in the group. These MS/MS results (including sequence data from the peptides) can be searched on in-house software or against the new NIH Mascot server. Our group has specialized in identifying the post-translational modifications in proteins. In particular, we have examined many proteins and characterized their disulfides. We also are working hard to develop methods for the characterization of glycosylation sites. N-linked glycoforms are easily removed with PGNaseF, and the Asn is changed to an Asp thereby leaving a marker. O-linked sites are lost after the glycosylation is removed. Our laboratory is working on a project to improve methods of positively identifying both N- and O-linked sites. We have a specific interest in not only identifying the sites, but also in determining all of the glyco-structures on these peptides. Work is underway to develope the techniques for this approach, including the development of software to aid in the interpretation. Other software on the laboratory web site at http://sx102a.niddk.nih.gov continues to be in heavy demand. As an increased understanding of protein interactions becomes known, the importance of isolating protein complexes and identifying the players is of big interest. The group is interacting with several research groups working in this area and developing the techniques to deal with these samples. Of particular interest is the identification of all the proteins in a specific proteome. The common approach is to digest an entire proteome with trypsin and then fractionate this by two dimensional chromatography. This method ?discards? all the chromatographic information as the entire 2-D liquid chromatographic run must be analyzed as one search set. We have chosen to pre-separate the proteins in the proteome using reverse phase (C18) chromatography and separately digest and characterize each fraction, as this should give a more concise and accurate search. After using some trial protein mixtures, we chose to use the proteome of Rickettsia. Reckettsia is a class B biowarfare agent and thus very relevant to NIH following the new initiatives here. Protein samples were supplied from the University of South Alabama and involved just the soluble proteins. Of the 834 proteins in the proteome, 53 were identified in the initial trial run using fast chromatography and limited data acquisition. This number expanded a little when longer chromatographic runs were tried but the method is far from optimized. However this ?proteome? methodology is far from optimized and will be the subject of further development work. For full details of the results so far, view the web site at http://sx102a.niddk.nih.gov/proteome.html. Other projects include: (1) The characterization of cyclized Green Fluorescent Protein (GFP), which is also being used as a tag for proteomic approaches. Cyclase Associated Protein, mutant cyanovirin and dihydrofolate reductase have been effectively characterized. (2) The characterization of the molecular structure of silk proteins. This required the development of methods to determine isotopically enriched proteins. (3) A similar method has been established to identify and characterize abnormal amino acids found in natural products targeted for their anti-HIV and anti-cancer activity. (4) We are working on methods to elucidate structural information of HIV-integrase cross-linked with DNA and Actinomyces naeslundii fimbriae. (5) In order to identify the most reactive sites in tubulin, every cysteine in the tubulin has been reacted with various sulfhydryl reagents. This method will be a very important tool in protein-drug binding studies as well as for understanding tubulin polymerization mechanism. (6) A method to characterize fluxes of palmitoylation of the TRH receptor is under investigation. This research is important to understand how and why post-translational modifications are triggered by environmental changes. The laboratory runs a large number of more rountine analyses, helping investigators from all over NIH. The demand for characterization of proteins has increased so dramatically that a new LCMS obtained from elsewhere in the Institute has been set up in our group for hands-on use by those investigators. The QTOF instrument, which was jointly purchased by five institutes, runs on an almost continuous basis.

现在，结构质谱组几乎完全集中在肽和蛋白质型样品上。这些可能作为凝胶上的斑点，重组蛋白样品的结构确认或翻译后修饰测定，作为蛋白质或蛋白质复合物沉淀或用抗体纯化的蛋白质或整个蛋白质组织。 凝胶上的斑点通常通过胰蛋白酶，还原和烷基化后的斑点消化，以及该组中QTOF质谱仪的随后分析。这些MS/MS结果（包括来自肽的序列数据）可以在内部软件或针对新的NIH Mascot服务器上进行搜索。我们的小组专门识别蛋白质的翻译后修饰。特别是，我们检查了许多蛋白质并表征了它们的二硫化物。我们还在努力开发用于表征糖基化位点的方法。使用PGNASEF很容易去除N连锁的糖衣，然后将ASN更改为ASP，从而留下标记。去除糖基化后，O连锁位点丢失。我们的实验室正在研究一个项目，以改善积极识别N-和O链接站点的方法。我们对不仅识别站点，而且对确定这些肽上的所有聚糖结构都有特定的兴趣。正在开展这种方法的技术，包括开发软件以帮助解释。 http://sx102a.niddk.nih.gov上的实验室网站上的其他软件继续满足。随着人们对蛋白质相互作用的越来越多的了解，隔离蛋白质复合物并识别玩家的重要性是引人注目的。该小组正在与在该领域工作的几个研究小组进行互动，并开发了处理这些样本的技术。 特别令人感兴趣的是特定蛋白质组中所有蛋白质的鉴定。常见的方法是用胰蛋白酶消化整个蛋白质组，然后通过二维色谱法分解它。这个方法？丢弃？必须将所有色谱信息作为整个二维液体色谱运行，必须分析为一个搜索集。我们选择使用反相（C18）色谱法预先分离蛋白质，并分别消化并表征每个分数，因为这应该给出更简洁，更准确的搜索。使用了一些试验蛋白混合物后，我们选择使用立克西亚的蛋白质组。 Reckettsia是B级BioWarfare代理商，因此与NIH非常相关，根据这里的新计划。从阿拉巴马大学提供蛋白质样品，仅参与可溶性蛋白质。在蛋白质组中的834种蛋白质中，使用快速色谱和有限的数据采集在初始试验中鉴定了53个蛋白质。当尝试了更长的色谱运行时，该数字会扩展一点，但该方法远非优化。但是这个？蛋白质组？方法论远非优化，将成为进一步发展工作的主题。有关结果到目前为止的完整详细信息，请在http://sx102a.niddk.nih.gov/proteome.html上查看网站。 其他项目包括：（1）环化绿色荧光蛋白（GFP）的表征，该蛋白质（GFP）也被用作蛋白质组学方法的标签。循环酶相关的蛋白质，突变体蓝黄素和二氢叶酸还原酶已有效地表征。 （2）丝蛋白分子结构的表征。这需要开发方法来确定同位素富集的蛋白质。 （3）已经建立了一种类似的方法来识别和表征针对抗HIV和抗癌活性的天然产物中发现的异常氨基酸。 （4）我们正在研究与DNA和放线症Naeslundii Fimbriae交联HIV - 积极酶结构信息的方法。 （5）为了鉴定微管蛋白中最具反应性的位点，小管蛋白中的每一种半胱氨酸都与各种硫酰酯试剂反应。该方法将是蛋白质毒物结合研究以及了解微管蛋白聚合机制的非常重要的工具。 （6）正在研究一种表征TRH受体棕榈酰化通量的方法。这项研究对于了解环境变化触发翻译后修饰的方式以及为什么是为何重要。 该实验室进行了大量的Rountine分析，帮助来自NIH的调查人员。对蛋白质表征的需求已大大增加，以至于我们小组中已经建立了从研究所其他地方获得的新LCM，以供这些研究者动手使用。由五家机构共同购买的QTOF仪器几乎连续运行。

项目成果

Nitric oxide-mediated heme oxidation and selective beta-globin nitrosation of hemoglobin from normal and sickle erythrocytes.

一氧化氮介导的血红素氧化和正常红细胞和镰状红细胞血红蛋白的选择性β-珠蛋白亚硝化。

• DOI: 10.1006/bbrc.2000.3413
• 发表时间: 2000
• 期刊: Biochemical and biophysical research communications
• 影响因子: 3.1
• 作者: Hrinczenko,BW;Schechter,AN;Wojtkowski,TL;Pannell,LK;Cashon,RE;Alayash,AI
• 通讯作者: Alayash,AI

Corydendramines A and B, defensive natural products of the marine hydroid Corydendrium parasiticum.

紫堇胺 A 和 B，海洋水螅寄生紫堇的防御性天然产物。

• DOI: 10.1021/np000050h
• 发表时间: 2000
• 期刊: Journal of natural products
• 影响因子: 5.1
• 作者: Lindquist,N;Shigematsu,N;Pannell,L
• 通讯作者: Pannell,L

Small molecular products of dealkylation in soman-inhibited electric eel acetylcholinesterase.

梭曼抑制的电鳗乙酰胆碱酯酶脱烷基化的小分子产物。

• DOI: 10.1021/bi991112
• 发表时间: 1999
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Viragh,C;Kovach,IM;Pannell,L
• 通讯作者: Pannell,L

Intermolecular aggregations are responsible for the slow kinetics observed in the folding of cytochrome c at neutral pH.

分子间聚集是造成细胞色素 c 在中性 pH 条件下折叠动力学缓慢的原因。

• DOI: 10.1006/jmbi.1999.3226
• 发表时间: 1999
• 期刊: Journal of molecular biology
• 影响因子: 5.6
• 作者: Nawrocki,JP;Chu,RA;Pannell,LK;Bai,Y
• 通讯作者: Bai,Y

Characterization of an enterotoxigenic Escherichia coli strain from Africa expressing a putative colonization factor.

来自非洲的表达推定定植因子的产肠毒素大肠杆菌菌株的表征。

• DOI: 10.1128/iai.67.8.4019-4026.1999
• 发表时间: 1999
• 期刊: Infection and immunity
• 影响因子: 3.1
• 作者: Khalil,SB;Cassels,FJ;Shaheen,HI;Pannell,LK;El-Ghorab,N;Kamal,K;Mansour,M;Savarino,SJ;PeruskiJr,LF
• 通讯作者: PeruskiJr,LF