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.

结构质谱组现在几乎完全集中在肽和蛋白质类型的样品。这些可以作为凝胶上的斑点、用于结构确认或翻译后修饰测定的重组蛋白样品、作为用抗体沉淀或纯化的蛋白质或蛋白质复合物或作为整个蛋白质组进入实验室。 在还原和烷基化后，通过用胰蛋白酶消化凝胶上的斑点，并随后在该组中的QTOF质谱仪上进行分析，常规鉴定凝胶上的斑点。这些MS/MS结果（包括肽的序列数据）可以在内部软件或新的NIH Mascot服务器上搜索。我们的团队专门从事蛋白质翻译后修饰的鉴定。特别是，我们已经研究了许多蛋白质，并确定了它们的二硫化物。我们也在努力开发糖基化位点的表征方法。N-连接的糖型很容易用PGNaseF去除，并且Asn变为Asp，从而留下标记。糖基化被去除后，O-连接位点丢失。我们的实验室正在进行一个项目，以改善积极识别N-和O-连接的网站的方法。我们有一个特定的兴趣，不仅在确定的网站，而且在确定所有的糖结构，这些肽。目前正在开发这种方法的技术，包括开发有助于解释的软件。实验室网站www.example.com上的其他软件需求量仍然很大。随着对蛋白质相互作用的了解越来越多，分离蛋白质复合物和识别参与者的重要性引起了人们的极大兴趣。该小组正在与从事这一领域工作的几个研究小组进行互动，并开发处理这些样本的技术。 特别感兴趣的是鉴定特定蛋白质组中的所有蛋白质。常用的方法是用胰蛋白酶消化整个蛋白质组，然后通过二维色谱对其进行分离。这个方法？丢弃物？作为整个二维液相色谱运行的所有色谱信息必须作为一个搜索集进行分析。我们选择使用反相（C18）色谱法预分离蛋白质组中的蛋白质，并分别消化和表征每个部分，因为这应该提供更简洁和准确的搜索。在使用一些试验蛋白质混合物后，我们选择使用立克次体的蛋白质组。雷克西亚是一种B类生物战剂，因此在这里的新举措之后与NIH非常相关。蛋白质样品由南亚拉巴马大学提供，仅涉及可溶性蛋白质。在蛋白质组中的834种蛋白质中，有53种是在最初的试验运行中使用快速色谱和有限的数据采集进行鉴定的。当尝试更长的色谱运行时，该数字略有扩大，但该方法远未优化。然而，这？蛋白质组？方法远未优化，将是进一步发展工作的主题。有关迄今为止结果的全部细节，请访问网站www.example.com。 其他项目包括：（1）环化绿色荧光蛋白（GFP）的表征，其也被用作蛋白质组学方法的标签。环化酶相关蛋白，突变体cyanovirin和二氢叶酸还原酶已被有效地表征。(2)丝蛋白分子结构的表征。这就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鉴定和表征天然产物中发现的异常氨基酸，这些天然产物具有抗HIV和抗癌活性。(4)我们正在研究方法，以阐明与DNA交联的HIV整合酶和内氏放线菌菌毛的结构信息。(5)为了鉴定微管蛋白中最具反应性的位点，微管蛋白中的每个半胱氨酸都与各种巯基试剂反应。这一方法将是一个非常重要的工具，在蛋白质-药物结合的研究，以及了解微管蛋白聚合机制。(6)一种表征TRH受体棕榈酰化通量的方法正在研究中。这项研究对于理解环境变化如何以及为什么会引发翻译后修饰非常重要。 该实验室进行了大量更常规的分析，帮助来自NIH各地的研究人员。对蛋白质表征的需求急剧增加，以至于从研究所其他地方获得的新的LCMS已经在我们组中建立，供这些研究人员亲自使用。QTOF仪器由五个研究所联合购买，几乎连续运行。

项目成果

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

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

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