Characterization Of Proteins By Mass Spectrometry

核仁蛋白B23/核磷蛋白。通过特定蛋白质与目标蛋白质的免疫共沉淀，可以检测到新的蛋白质-蛋白质相互作用。我们分析了非洲爪哇卵提取物中与Ulp1p类相扑酶xSENP3和5特异结合的蛋白质。通过胰酶消化和LC/MS/MS分析凝胶分离的蛋白质，可以鉴定出几个新的结合伙伴。一种与xSENP3强结合的蛋白质被鉴定为非洲爪哇B23/核磷蛋白同源物。进一步的实验(在LGRD进行)表明，这种结合对于SENP3和SENP5在哺乳动物组织培养细胞中的稳定积累是必不可少的。 血清中脂质的定量。我们一直在继续开发用质谱仪测定人血清中心磷脂的方法学。这一努力与该研究所正在进行的一项临床研究有关，该研究旨在评估感染B组链球菌(GBS)生物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。在对新生绵羊的其他研究中已经证明，在青霉素治疗下，GBS生物体分泌一种特殊的细胞壁膜心磷脂，这种物质在血清中的水平约为100 pmole/ml时会导致呼吸窘迫。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。该分析方法包括在200ul血清样本中加入内标，液-液和固相萃取相结合，然后进行LC-MS分析，其中包括提取/回收标准，以监测系统质量控制。我们已经证明，从血清中提取心磷脂的效率高于75%，并且可以测定低于100pmole/mL的血清心磷脂水平。我们继续改进测定这些物质的质谱学方法，以提高分析的稳健性和样品吞吐速度。 微管蛋白翻译后修饰。最近，关于微管蛋白翻译后修饰的持续工作在磷酸化位点方面产生了有趣的结果。在鸡红细胞微管蛋白C端的CNBR酶切片段上添加了80Da的特征片段，表明存在磷酸化，但到目前为止还没有鉴定到修饰的残基。在另一项研究中，使用了一种已知的在2摩尔磷酸盐与摩尔微管蛋白比率中磷酸化微管蛋白的Kinase，已经确定了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-微管蛋白被证明是磷酸化的，不是在C-末端区域，而是在蛋白质的主序列中，X射线结晶学已知的区域存在于原丝外的一个环中，该环在微管组装中发挥积极作用。然而，到目前为止，还没有鉴定出α-微管蛋白上的类似位点。这一观察可能具有实质性的治疗意义，特别是考虑到在阿尔法微管蛋白的类似区域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部位。 频谱可靠性的改进。通过从多个重复中产生共识谱来开发方法以获得特定样品的更可靠的质谱图的努力继续卓有成效。作为推断凝胶样品中蛋白质存在的努力的一部分，当我们将其应用于肽裂解光谱时，我们的初步结果显示，通过使用这种方法，结果有了实质性的改善。当应用于肽序列从头测序时，这种方法的效果尤其显著。虽然我们的新算法用于推导从头开始的多肽序列通常会对特定的多肽前体片段产生有用的结果，但从同一多肽前体的多个复制光谱中生成一致的光谱似乎每次都会给出有意义的结果。受到这些结果的鼓舞，我们计划将这种方法扩展到MALDI TOF-TOF和LC/MS-MS方法中从未知样本进行常规蛋白质推断的方法。

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.