Characterization Of Proteins and Other Molecules By Mass Spectrometry

尼曼-皮克病C型(NPC)的脑蛋白质组学研究我们研究了5周龄对照小鼠(n=7)和NPC1突变雌性小鼠(n=7)的小脑组织。对四倍体凝胶上的871个蛋白质点进行统计分析后发现，NPC1-/-小脑组织中共有52个蛋白质点显著增加(R&#8805；1.20，p&lt；0.05)，12个蛋白质点显著减少(R&#8804；0.83，p&lt；0.05)。从成对的突变体和对照制备凝胶中分离出差异表达的蛋白质点，并使用质谱学技术对20对蛋白质点进行了候选鉴定。尽管验证还在进行中，但其中一种已确定的蛋白质是转甲状腺蛋白。虽然这种蛋白质的鉴定是在小鼠脑中进行的，但转甲状腺激素在人类脑脊液中是一个有趣的潜在生物标记物。其中一个原因是，脑脊液转甲状腺素水平的降低与阿尔茨海默病的疾病严重程度相关。这些研究现在已经扩展到一周和三周大的仔猪和总共200个额外的差异表达凝胶点。 鼻咽癌患者的脑脊液分析。初步实验表明，使用我们新的分析方法来比较蛋白质谱，我们可以检测到鼻咽癌患者和成人对照组的脑脊液之间的显著差异。脑脊液样本采集自37例鼻咽癌患者。目前为鼻咽癌患者收集的样本包括2.5年以来收集的纵向样本和5对米非司汀前后的样本。此外，儿童国家医疗中心正在收集儿科脑脊液样本。我们的分析方法考虑了系统的方差来源，对MALDI-TOF谱集合使用了方差分析(ANOVA)方法，然后对由ANOVA生成的数据阵列应用了主成分分析。ANOVA-PCA分析的结果将使用神经严重程度量表与患者的疾病状况相关联，该量表可用于定义SMD、NICHD开发的鼻咽癌患者的疾病状况和进展。正在使用模糊多变量规则构建专家系统和最小神经网络(FIRS)的数学工具进一步分析广泛的差异观测剖面谱，以阐明潜在的特定生物标志物。这项分析的初步结果显示，至少存在10个特定的多肽团块，它们是候选生物标记。我们将使用脑脊液的色层分离和质谱仪来鉴定脑脊液中的特定多肽，这是Fures分析的95%可信区间所建议的。 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，GBS生物在青霉素治疗下分泌一种特定的细胞壁膜心磷脂，这种物质导致呼吸窘迫的水平相当于在血清中注射约100 pmole/ml；注意，这种浓度迅速下降，半衰期为几分钟。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。分析方法包括在血清样本中加入内标，液-液和固相结合进行提取，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约为90%。我们进一步表明，正常成人水平的(18：2)4心磷脂存在于血清中的水平&lt；10fmole/ul，比先前不太准确的测量结果大约低1000倍。有了这一基线信息，我们正在寻求建立已知未被GBS定植的母亲脐带血中的正常血清水平，然后将确定感染个体的水平。

Brain Proteomics in Niemann-Pick Disease- Type C (NPC). We have characterized cerebellar tissue from five weeks old littermate control (n=7) and Npc1 mutant female mice (Npc1m1N, n=7). After statistical analysis of 871 protein spots on quadruplicate gels, a total of 52 protein spots were found to be significantly increased (R&#8805;1.20, p<0.05) and 12 protein spots significantly decreased (R&#8804;0.83, p<0.05) in Npc1-/- cerebellar tissue. Differentially expressed protein spots were isolated from paired mutant and control preparative gels, and using mass spectrometric techniques we have candidate identifications, to date, on 20 pairs of protein spots. Although validation is pending, one of the identified proteins is transthyretin. Although this protein identification was made in mouse brain, transthyretin is an intriguing potential biomarker in human CSF. One reason for this is that decreased levels of cerebral spinal fluid transthyretin correlate with disease severity in Alzheimer disease. These studies have now been extended to one and three weeks old littermates and a total of 200 additional differentially expressed gel spots. Cerebral Spinal Fluid Profiling (CSF) in NPC. Initial experiments have demonstrated that we can detect significant differences between cerebral spinal fluid from NPC patients and adult controls using our novel analytical approach to compare protein profiles. CSF samples have been collected from 37 well-characterized NPC patients. Currently the collection for NPC patients includes longitudinal samples collected over 2.5 years and five pre/post miglustat pairs. In addition a collection of pediatric CSF samples is in progress at Childrens National Medical Center. Our analytical approach accounts for systemic sources of variance, employs an analysis of variance (ANOVA) approach to the sets of MALDI-TOF spectra, and then applies Principal Component Analysis to the data arrays generated by ANOVA. The results of ANOVA-PCA analyses will be correlated with patient disease status using a neurological severity scale that can be used to define disease status and progression in NPC patients as developed by the SMD, NICHD. The extensive differentially observed profile spectra are being analyzed further using mathematical tools of Fuzzy multivariate rule-building expert systemsminimal neural networks (FuRES) to elucidate potential specific biomarkers. Initial results of this analysis show the presence of at least 10 specific peptide masses that are candidate biomarkers. We will employ chromatographic separation of the CSF followed by mass spectrometry to identify specific peptides in the CSF suggested by the 95% Confidence Intervals of the FuRES analysis. 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 corresponding to the injection of about 100 pmole/mL in serum; note that this concentration falls rapidly with a half life of a few minutes. 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 serum sample, extractions by a combination of liquid-liquid and solid phase, 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 approximately 90% efficiency. We have further shown that normal adult levels of (18:2)4 cardiolipins are present in serum at levels < 10 fmole/uL, approximately 1000-fold lower than found by earlier, less accurate measurements. With this base line information, we are seeking to establish normal serum levels in cord blood of mothers known to be uncolonized by GBS and will then determine levels in infected individuals.