Characterization Of Proteins By Mass Spectrometry

核仁蛋白B23/核磷蛋白。新的蛋白质-蛋白质相互作用可以通过特异性蛋白质与感兴趣的靶蛋白质的免疫共沉淀来检测。我们分析了爪蟾卵提取物中特异性结合Ulp 1 p样SUMO蛋白酶xSENP 3和5的蛋白质。 通过胰蛋白酶消化，然后通过LC/MS/MS分析凝胶分离的蛋白质，可以鉴定几种新的结合伴侣。 与xSENP 3强烈结合的蛋白质被鉴定为非洲爪蟾B23/核磷蛋白同源物。通过进一步的实验（在LGRD中进行）显示，这种结合对于SENP 3和SENP 5在哺乳动物组织培养细胞中的稳定积累是必需的。 血清中的脂质定量。我们一直在开发通过质谱法定量人血清中心磷脂的方法。这一努力与该研究所正在进行的一项临床研究有关，该研究旨在评估抗生素治疗感染B组链球菌（GBS）微生物的孕妇的效果。该研究的假设是，典型的围产期青霉素治疗引起婴儿循环心磷脂大量增加，然后导致呼吸窘迫。在新生绵羊的其他研究中已经证明，GBS微生物在青霉素治疗下分泌特定的细胞壁膜心磷脂，并且该物质在血清中约100 pmole/mL的水平下引起呼吸窘迫。目前尚不清楚在GBS定植母亲所生婴儿中观察到的呼吸窘迫是否是类似效应的结果，或者可能是细菌死亡刺激内源性心磷脂释放引起的相关效应。该分析方法包括向200 uL血清样品中加入内标物，结合液-液和固相萃取，然后进行LC-MS分析，其中包含萃取/回收标准品以监测系统质量控制。我们已经表明，心磷脂可以从血清中提取，效率大于75%，心磷脂血清水平低于100 pmole/mL可以确定。我们继续完善用于测定这些材料的质谱方法，以提高分析的稳健性和样品通量。 微管蛋白翻译后修饰。关于微管蛋白翻译后修饰的持续研究最近在磷酸化位点方面取得了有趣的结果。一个80 Da的鸡红细胞微管蛋白的C-末端溴化氰消化片段的特征除了显示磷酸化的存在下，但迄今为止，修改后的残基尚未确定。在另一项研究中，使用已知以2摩尔磷酸盐与摩尔微管蛋白的比率磷酸化微管蛋白的激酶，已经鉴定了HeLa微管蛋白的磷酸化位点。在HeLa细胞中发现的两种不同的β-微管蛋白已被证明磷酸化，不是在C-末端区域，而是在蛋白质的主要序列中，该区域通过X射线晶体学已知存在于原丝外部的环中，其在微管组装中起着积极的作用。然而，在这一点上，α-微管蛋白上的类似位点尚未被鉴定。这一观察结果可能具有实质性的治疗意义，特别是鉴于α微管蛋白的类似区域中存在或不存在磷酸化，因为已知该环是他莫昔芬抑制微管组装的位点。 光谱可靠性的提高。在开发方法以通过从多个重复样品中生成共有谱来获得给定样品的更稳健的质谱方面的努力继续是富有成效的。当应用于肽片段化谱作为推断凝胶样品中蛋白质存在的努力的一部分时，我们有初步的结果显示，通过使用这种方法，结果有了实质性的改善。当应用于肽从头测序时，这种方法的效果特别显著。虽然我们用于从头推导肽序列的新算法通常会为被片段化的特定肽前体产生有用的结果，但从相同肽前体的多个重复光谱生成共识光谱似乎每次都能给出有意义的结果。受这些结果的鼓舞，我们计划将该方法扩展到MALDI TOF-TOF和LC/MS-MS方法中未知样品的常规蛋白质推断。

Nucleolar protein B23/Nucleophosmin. Novel protein-protein interactions can be detected by co-immunoprecipitation of specific proteins with a target protein of interest. We analyzed proteins from Xenopus egg extracts that specifically bound to the Ulp1p-like SUMO proteases xSENP3 and 5. Analysis of gel separated proteins by tryptic digestion followed by LC/MS/MS allowed identification of several novel binding partners. A protein that strongly bound to xSENP3 was identified as the Xenopus B23/Nucleophosmin homolog. This binding was shown by further experiments (carried out in LGRD) to be essential for stable accumulation of SENP3 and SENP5 in mammalian tissue culture cells. Lipid Quantification in Serum. We have continued developing methodology to quantify cardiolipins in human serum by mass spectrometry. This effort is in association with a clinical study underway in the Institute to evaluate the effects of antibiotic treatment of pregnant women colonized with the Group B streptococcal (GBS) organism. 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 newborn sheep that the GBS organisms secrete a specific cell wall membrane cardiolipin with penicillin treatment and that this substance causes respiratory distress at levels of about 100 pmole/mL in serum. 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. The analytical approach involves the addition of an internal standard to a 200 uL serum sample, a combination of liquid-liquid and solid phase extractions, followed by an LC-MS analysis that incorporates an extraction/recovery standard to monitor system quality control. We have shown that cardiolipin can be extracted from serum with greater than 75% efficiency and that cardiolipin serum levels of less than 100 pmole/mL can be determined. We continue to refine the mass spectrometric methods for the determination of these materials in order to increase analytical robustness and sample throughput rate. Tubulin Post-translational Modifications. Continuing work on characterization of post-translational modifications of tubulins has recently yielded interesting results with respect to phosphorylation sites. A characteristic addition of 80Da to a chicken erythrocyte tubulin C-terminal CNBr digest fragment shows the presence of phosphorylation, but to date the modified residue has not been identified. In another study employing a kinease known to phosphorylate tubulin in a 2 mole phosphate to mole tubulin ratio, phosphorylation sites of HeLa tubulins have been identified. Two different beta-tubulins found in HeLa cells have been shown to phosphorylate, not in the C-terminal region, but in the main sequence of the proteins in a region known by X-ray crystallography to exist in a loop, external to the protofilaments, that plays an active role in microtubule assembly. To this point however, analogous sites on alpha-tubulins have not been identified. There may be substantial therapeutic implications to this observation, particularly in light of the presence or absence of phosphorylation in the similar region of the alpha tubulins, since this loop is known to be the site in which tamoxifen inhibits microtubule assembly. Improvement in Spectral Reliability. The efforts in development of methods to obtain more robust mass spectra of given samples by generating consensus spectra from multiple replicates have continued to be productive. When applied to peptide fragmentation spectra as part of an effort to infer the presence of proteins in gel samples, we have preliminary results showing a substantial improvement in results by using this approach. The effect of this approach is particularly significant when applied to peptide sequencing de novo. While our novel algorithm for deducing peptide sequences de novo often yields useful results for a particular peptide precursor being fragmented, generating a consensus spectrum from multiple replicate spectra of the same peptide precursor seems to give meaningful results every time. Encouraged by these results, we plan on extending the approach to routine protein inference from unknown samples in both MALDI TOF-TOF and LC/MS-MS methodologies.