权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Characterization Of Proteins By Mass Spectrometry

通过质谱法表征蛋白质

基本信息

批准号：
7734734
负责人：
ALFRED L YERGEY
金额：
$ 72.29万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
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/7734734
关键词：
Algorithms Amino Acid Sequence Antibiotic Therapy Binding C-terminal Cardiolipins Cell Wall Cessation of life Characteristics Chickens Clinical Research Co-Immunoprecipitations Consensus Cyanogen Bromide Digestion Endopeptidases Erythrocytes Gel Hela Cells Homologous Gene Human Infant Institutes Light Lipids Liquid substance Mass Spectrum Analysis Measurement Melanocytic nevus Membrane Methodology Methods Microtubules Mole the mammal Molecular Weight Monitor Mothers Newborn Infant Organism Penicillins Peptide Hydrolases Peptide Sequence Determination Peptides Phase Phosphorylation Phosphorylation Site Play Post-Translational Protein Processing Pregnant Women Proteins Quality Control Rate Recovery Respiratory distress Role Sampling Serum Sheep Site Solid Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Standards of Weights and Measures System Tamoxifen Therapeutic Time Tubulin Work X-Ray Crystallography Xenopus Xenopus Proteins alpha Tubulin beta Tubulin egg inorganic phosphate interest method development novel nucleophosmin protein aminoacid sequence protein protein interaction research study tissue/cell culture

项目摘要

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.

核仁蛋白 B23/核磷蛋白。新型蛋白质-蛋白质相互作用可以通过特定蛋白质与感兴趣的靶蛋白的免疫共沉淀来检测。我们分析了非洲爪蟾卵提取物中与 Ulp1p 样 SUMO 蛋白酶 xSENP3 和 5 特异性结合的蛋白质。通过胰蛋白酶消化和 LC/MS/MS 分析凝胶分离的蛋白质，从而鉴定出几种新型结合伴侣。与 xSENP3 强烈结合的蛋白质被鉴定为爪蟾 B23/核磷蛋白同源物。进一步的实验（在 LGRD 中进行）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至关重要。血清中的脂质定量。我们继续开发通过质谱法定量人血清中心磷脂的方法。这项工作与该研究所正在进行的一项临床研究相关，该研究旨在评估抗生素治疗感染 B 族链球菌 (GBS) 的孕妇的效果。该研究的假设是，典型的围产期青霉素治疗会导致婴儿循环心磷脂大量增加，从而导致呼吸窘迫。其他针对新生羊的研究表明，GBS 生物体在接受青霉素治疗后会分泌一种特定的细胞壁膜心磷脂，这种物质在血清中浓度约为 100 pmole/mL 时会引起呼吸窘迫。目前尚不清楚 GBS 定植母亲所生的一小部分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。分析方法包括向 200 uL 血清样品中添加内标，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。我们已经证明，可以以大于 75% 的效率从血清中提取心磷脂，并且可以测定低于 100 pmole/mL 的心磷脂血清水平。我们不断完善测定这些材料的质谱方法，以提高分析稳健性和样品通量。微管蛋白翻译后修饰。最近对微管蛋白翻译后修饰特征的持续研究在磷酸化位点方面取得了有趣的结果。鸡红细胞微管蛋白 C 端 CNBr 消化片段中特征性添加 80Da 显示磷酸化的存在，但迄今为止尚未鉴定出修饰残基。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，已鉴定出 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-微管蛋白已被证明不是在 C 末端区域磷酸化，而是在 X 射线晶体学已知的一个区域的蛋白质主序列中磷酸化，该区域存在于原丝外部的环中，在微管组装中发挥着积极作用。然而到目前为止，α-微管蛋白上的类似位点尚未被发现。这一观察结果可能具有重要的治疗意义，特别是考虑到α微管蛋白的相似区域是否存在磷酸化，因为已知该环是他莫昔芬抑制微管组装的位点。提高光谱可靠性。通过从多个重复中生成共有光谱来开发给定样品的更可靠的质谱的方法的努力一直富有成效。当应用于肽碎片光谱作为推断凝胶样品中蛋白质存在的努力的一部分时，我们的初步结果显示使用这种方法的结果得到了显着改善。当应用于肽从头测序时，这种方法的效果尤其显着。虽然我们用于从头推导肽序列的新算法通常会对被片段化的特定肽前体产生有用的结果，但从同一肽前体的多个重复光谱生成一致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 方法中从未知样品进行常规蛋白质推断。