Mass Spectrometry Of Drugs, Natural Products, Proteins,

结构质谱组现在几乎完全集中在多肽和蛋白质类型的样品上。这些蛋白质可以作为凝胶上的斑点、用于结构确认或翻译后修饰确定的重组蛋白质样本、作为与抗体沉淀或纯化的蛋白质或蛋白质复合体，或者作为完整的蛋白质组来进入实验室。 凝胶上的斑点通常是通过胰酶消化斑点，经过还原和烷基化，然后在QTOF质谱仪上进行分析来鉴定的。这些MS/MS结果(包括多肽的序列数据)可以在内部软件上搜索，也可以根据新的NIH Mascot服务器进行搜索。我们的团队专门研究蛋白质的翻译后修饰。特别是，我们研究了许多蛋白质，并对它们的二硫键进行了表征。我们还在努力开发表征糖基化位点的方法。N-连接的糖形式很容易被PGNaseF去除，并且AsN被改变为Asp，从而留下标记。糖基化被去除后，O-连接的位点就会丢失。我们的实验室正在致力于一个项目，以改进积极识别N-和O-连接位点的方法。我们特别感兴趣的不仅是确定这些位点，而且还包括确定这些多肽上的所有糖结构。目前正在努力开发这一方法的技术，包括开发协助口译的软件。Http://sx102a.niddk.nih.gov实验室网站上的其他软件需求依然旺盛。随着对蛋白质相互作用了解的加深，分离蛋白质复合体和识别参与者的重要性引起了人们的极大兴趣。该小组正在与这一领域的几个研究小组进行互动，并开发处理这些样本的技术。 特别令人感兴趣的是鉴定特定蛋白质组中的所有蛋白质。通常的方法是用胰酶消化整个蛋白质组，然后用二维层析法进行分离。这种方法？丢弃？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。我们选择了用反相(C18)层析预先分离蛋白质组中的蛋白质，并分别消化和鉴定每个组分，因为这应该会给出一个更简洁和准确的搜索。在使用了一些试验的蛋白质混合物后，我们选择使用立克次体的蛋白质组。Reckettsia是一种B类生物制剂，因此在这里的新举措之后与NIH非常相关。蛋白质样本由南阿拉巴马大学提供，只涉及可溶性蛋白质。在蛋白质组中的834种蛋白质中，有53种在最初的试运行中使用快速层析和有限的数据采集进行了鉴定。当尝试更长的层析运行时间时，这个数字略有增加，但方法远未达到最优化。但是这个呢？蛋白质组吗？方法论还远远没有得到优化，将是进一步开发工作的主题。有关到目前为止的结果的全部细节，请访问网站：http://sx102a.niddk.nih.gov/proteome.html. 其他项目包括：(1)环化绿色荧光蛋白(GFP)的表征，它也被用作蛋白质组学方法的标签。环化酶相关蛋白、突变型氰维菌素和二氢叶酸还原酶已被有效地鉴定。(2)蚕丝蛋白的分子结构表征。这就需要开发确定同位素浓缩蛋白质的方法。(3)已经建立了一种类似的方法来鉴定和表征天然产品中发现的针对其抗艾滋病毒和抗癌活性的异常氨基酸。(4)我们正在研究与DNA和奈氏放线菌菌毛交联的HIV整合酶的结构信息。(5)为了确定微管蛋白中活性最强的部位，微管蛋白中的每个半胱氨酸都与不同的巯基试剂反应。该方法将成为研究蛋白质-药物结合以及了解微管蛋白聚合机理的重要工具。(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触发翻译后修改是很重要的。 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。对蛋白质特性的需求急剧增加，以至于我们的小组已经建立了一个从研究所其他地方获得的新的LC MS，供这些研究人员动手使用。QTOF仪器由五个研究所联合购买，几乎是连续运行的。

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.