Characterization Of Proteins By Mass Spectrometry

为了提高蛋白质表征能力，正在进行几个领域的开发。首先，我们开发了一种新的方法来提供由于数据库错误、剪接变体或SNPs而未在数据库中描述的蛋白质的序列信息；这种不完整性最常与具有未知或部分特征的基因组的生物体有关，例如莱氏X.laevis。我们正在采取的方法，我们已被称为德新肽测序，通过穷尽列举的多肽成分，EEPC。我们最近使用一种改进的算法修改了这种方法，该算法使用图论方法来解决这个问题，使我们能够将LIPCUT数据库的大小限制在500Da范围内，LIPCUT数据库是所有肽质量组合的详尽列表。这使计算速度提高了10倍。我们还扩展了程序的能力，不仅考虑到观察到的光谱中的内部片段，而且增加了包括正在测序的多肽的许多翻译后修饰的能力。NIST的一个实验室正在对该方法进行独立评估。 在一个与蛋白质鉴定和测序相关的领域，我们正在研究微管蛋白的C末端翻译后修饰。除了之前报道的样品制备进展，在过去的一年里，我们开发了一种从多次重复测量中开发可靠光谱的方法。我们的方法是基于共识谱的形成，并包括从多个重复计算平均中心谱。我们改进了现有的文库形成方法，认识到当矢量点乘积用于评估相似性时，可以通过使用Fisher Z变换在95%的置信度水平上赋予统计置信度。这种方法的使用使我们能够对大鼠和牛脑微管蛋白的翻译后修饰模式的微管蛋白同型表达进行强有力的比较。此外，这种方法在一般意义上适用于任何质谱学数据，它的使用应导致为若干目的产生更可靠的光谱。在这一点上，该方法已被应用于复制多肽碎片谱，并已证明其使用导致从所得到的共识谱中更可靠地确定从头开始的序列。 在一个与蛋白质翻译后修饰相关的领域，我们开发了一种量化位点特异性酪氨酸磷酸化的方法。利用电喷雾电离质谱仪中激活的高亲和力IgE受体模型系统，利用一套外部多肽标准品进行了定量。肥大细胞上高亲和力的IgE受体与IgE结合是启动炎症反应的信号通路的第一步。已有研究表明，这种激活需要蛋白酪氨酸激酶的Src家族，但详细的分子机制尚未阐明。IgE受体的伽马亚基在羧基末端附近含有两个酪氨酸残基，它们是潜在的磷酸化位点。每种酪氨酸的定点突变表明，这两种酪氨酸都可以作为磷酸化的底物。根据在胰酶消化中观察到的磷酸肽，在适当的位置与磷酸酪氨酸或酪氨酸合成多肽。用两个候选区域对应的合成肽生成标准曲线来计算磷酸酪氨酸和酪氨酸的相对电离响应，并用来计算每个位点的磷酸酪氨酸的化学计量比。使用每对标准的标准曲线，并测量每个候选酪氨酸残基的磷酸化与未修饰肽的比率，结果表明，在激活的IgE受体伽马亚基中，Tyr65的磷酸化水平明显高于Tyr76。虽然用于这个特定的系统，显然该方法是一个通用的方法，可以广泛用于表征磷酸化化学计量学。 在另一个与蛋白质无关的领域，我们一直在开发通过质谱学来定量人体血清中心磷脂的方法。这一努力的目的是与该研究所正在开展的一项临床研究相联系，该研究旨在评估感染B组链球菌(GBS)的孕妇接受抗生素治疗的效果。这项研究的假设是，典型的围产期青霉素治疗会导致婴儿循环中的心磷脂大量增加，从而导致呼吸窘迫。在对胚胎绵羊的其他研究中已经证明，在青霉素治疗下，GBS生物体分泌一种特定的细胞壁膜心磷脂，这种物质会导致绵羊的呼吸窘迫。目前尚不清楚在GBS定居母亲所生婴儿中观察到的呼吸窘迫是类似效应的结果，还是细菌死亡刺激内源性心磷脂释放所引起的相关效应。考虑到分娩时可从婴儿脐带血中获得的样本数量有限，我们已经开发出适用于少量(约20微升)血清的心磷脂提取程序。我们已经制定了适当的量化标准，与标准添加方法相结合，将允许量化。此外，我们还开发了一种将该标准引入血清样本的可靠方法，并表明可以以大约75%的效率从血清中提取心磷脂。目前，我们正在完善测定这些物质的质谱学方法。

There are several areas of development that are being followed in order to improve protein characterization capabilities. First, we have developed a novel approach to providing sequence information for proteins that are not described in data bases, due to data base error, incompleteness splice variants or SNPs; this incompleteness is associated most frequently with organisms having unknown or partially characterized genomes, e.g. X. laevis. We are taking the approach we have termed De Novo Peptide Sequencing through Exhaustive Enumeration of Peptide Compositions, EEPC. We have recently modified this approach using an improved algorithm that uses a graph theory approach to the solution of the problem enabling us to limit the size of the LIPCUT data base, the exhaustive listing of all peptide mass combinations, to a 500 Da range. This has increased the computational speed 10-fold. We have also extended the capability of the program to account not only for internal fragments in the observed spectra, but we have added the capability to include many post-translational modifications of the peptides being sequenced. The methodology is being evaluated independently by a laboratory at NIST. In a area related to protein identification and sequencing, we are characterizing the C-terminal post-translational modifications of tubulins. In addition to the sample preparation advances that were reported previously, in the past year we have developed a means of developing reliable spectra from multiple replicate measurements. Our approach is based on the formation of a consensus spectrum and consists of calculating a mean centered spectrum from multiple replicates. We have improved upon existing library formation methods by the recognition that a vector dot product, when used to assess similarity, can be assigned a statistical confidence at a 95% confidence level with the use of the Fisher Z-transform. The use of this approach has enabled us to make a robust comparison of tubulin isotoype expression an posttranslational modification patterns in rat and cow brain tubulin. In addition, this approach is applicable in a general sense to any mass spectral data and its use should result in the generation of more reliable spectra for a number of purposes. In this light the approach has been applied to replicate peptide fragmentation spectra and has demonstrated that its use leads to more reliable determination of de novo sequences from the resultant consensus spectrum. In an area related to characterization of protein posttranslational modifications, we have developed a methodology for the quantification of site specific tyrosine phosphorylation. Using the model system of activated high affinity IgE receptors in an electrospray ionization mass spectrometer, the quantification has been carried out using a set of external peptide standards. The binding of IgE to high affinity IgE receptors on mast cells is the initial step in activation of signaling pathways that initiate an inflammatory response. It has been demonstrated that the Src family of protein tyrosine kinases are required for this activation, however the detailed molecular mechanism has not been elucidated. The gamma subunit of the IgE receptor contains two tyrosine residues near the carboxyl terminus that are potential sites for phosphorylation. Site directed mutation of each tyrosine demonstrated that both tyrosines can be substrates for phosphorylation. Based on the phosphopeptides observed in tryptic digests, peptides were synthesized with either phospho-Tyr or Tyr at the appropriate sites. Synthetic peptides corresponding to both of the two candidate regions were used to generate standard curves to calculate relative ionization responses for phospho-Tyr and Tyr, and used to calculate the stoichiometry for phospho-Tyr at each site. Using standard curves for each pair of standards, and measuring the ratio of phosphorylated to unmodified peptide for each of the candidate tyrosine residues, it was demonstrated that Tyr65 was phosphorylated at significantly higher level than Tyr76 in the activated IgE receptor gamma subunit. While used for this specific system, clearly the approach is a general one and could be used widely to characterize phosphorylation stoichiometry. In another area, unrelated to proteins, we have been developing methodology to quantify cardiolipins in human serum by mass spectrometry. The purpose of this effort is in association with a clinical study being developed by the Institute to evaluate the effects of antibiotic treatment of pregnant women with Group B streptococcal (GBS)infections. The hypothesis of the study is that the typical peri-natal penicillin treatment gives rise to a large increase of circulating cardiolipins in the infant which then leads to respiratory distress. It has been demonstrated in other studies in fetal sheep that the GBS organisms secrete a specific cell wall membrane cardiolipin with penicillin treatment and that this substance causes respiratory distress in the sheep. It is not known whether the respiratory distress observed in a fraction of infants born to GBS colonized mothers is a result of a similar effect, or perhaps by a related effect caused by a release of endogenous cardiolipins stimulated by the bacterial death. We have developed cardiolipin extraction procedures for small quantities, ca. 20 uL, of serum in recognition of the limited sample sizes that will be available from infant cord blood at delivery. We have developed appropriate quantification standards that in conjunction with a standard additions approach will allow for quantification. In addition, we have developed a reliable method for introducing this standard into a serum sample and have shown that cardiolipin can be extracted from serum with about a 75% efficiency. At present we are refining the mass spectrometric methods for the determination of these materials.