Characterization Of Proteins By Mass Spectrometry

Smith-Lemli-Opitz综合征(SLOS)的脑蛋白质组学研究这项研究使用了涉及胆固醇生物合成缺陷的人类遗传病的小鼠模型。通过对野生型和基因敲除小鼠(Dhcr7和Sc5d)脑组织的2D凝胶蛋白质组学研究，确定了蛋白的表达差异，通过三重2D凝胶的图像分析确定了差异超过1.5倍。对于蛋白质鉴定，至少有3个肽被匹配，置信度大于95%，总氨基酸序列覆盖率从总蛋白质的14%到74%。HMG辅酶A合成酶是在脑提取液中观察到的差异表达蛋白质的一个例子。与野生型相比，基因敲除动物脑组织中的HMG CoA合成酶显著增加，Dhcr7和Sc5d的对照和敲除比例分别为1：1.55和1：1.75。由于HMG辅酶A合成酶是胆固醇生物合成途径的第一步，大脑胆固醇水平的降低有望增加该酶的活性/表达，这在两个基因敲除中都观察到了。除了蛋白质表达水平的变化外，该方法还检测到基因敲除小鼠大脑蛋白质中蛋白质翻译后修饰的变化。这些修饰是从凝胶上蛋白质迁移率的变化中检测到的。观察到Cofilin 1的这种变化的一个例子。观察到两个含有Cofilin 1的凝胶斑点，并且观察到与野生型对照相比，在基因敲除小鼠中具有更酸性等电点的斑点具有更强的迁移强度。Western blotts用一种抗Cofilin 1抗体证实了Cofilin 1磷酸化的增加。另一个蛋白质迁移转变的例子是Rab7蛋白质。RAB 7是GTP结合蛋白大家族中的一员。这个家族中的许多蛋白质在C-末端通过半胱氨酸残基的戊烯基化修饰。蛋白质预烯基化是胆固醇生物合成生化途径中的一个分支，胆固醇生物合成的改变也会导致蛋白质预烯基化水平的改变。已知预烯化会改变至少一些GTP结合蛋白的迁移率，数据表明Rab7预烯基化的变化可能会导致观察到的迁移率改变。然而，在这里没有直接确定Rab7的前烯基化，也可能涉及其他翻译后修饰。 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，正常成人的(18：2)4心磷脂水平存在于血清中，水平为10 fmole/ul，比先前不太准确的测量结果低约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。 组蛋白去甲基化的量化。组蛋白甲基化调节细胞的染色质结构、转录和表观遗传状态。组蛋白甲基化受组蛋白甲基酶和去甲基酶的动态调节，组蛋白甲基酶和去甲基酶介导组蛋白二甲基化和单甲基化。目前还不清楚是否存在逆转赖氨酸三甲基化的去甲基酶。在早期的研究中发现了一种可能的组蛋白去甲基酶KDM3B，它正在被研究，以探索其去甲基化能力的特异性和程度，使用含有1，2或3个甲基化的赖氨酸残基合成的模型肽。将甲基化多肽ARTKQTARKSTGGKAPRKQLAGGK生物素与免疫沉淀(IP)或重组KDM3B孵育，用MALDI/TOF测定去甲基化产物与底物的比例。重复来自对照和酶实验的多肽底物的质谱测量，无论有没有假定的辅助因子RanQ69L-GTP，都根据背景水平进行校正，并计算部分去甲基化。重组KDM3B的初步研究表明没有活性，而IP酶显示了1-Me的脱甲基化活性、2-Me上两个甲基的去除以及3-Me底物上的NO活性。

Brain Proteomics in Smith-Lemli-Opitz Syndrome (SLOS). This investigation uses mouse models for human genetic diseases involving defects in cholesterol biosynthesis. 2D gel-based proteomic studies of wild type and knockout mice (Dhcr7 and Sc5d) brain tissue have been led to the determination of 64 proteins with expression differences exceeding 1.5-fold as determined from image analysis of triplicate 2D gels. For protein identification, at least 3 peptides were matched with greater than 95% confidence, and total amino acid sequence coverage ranged from 14% to 74% of the total protein. HMG CoA synthase is an example of a differentially expressed protein observed in brain extracts. HMG CoA synthase in brains of knockout animals was significantly increased relative to wild type, with a ratio of control to knockout equal to 1:1.55 for Dhcr7, and 1:1.75 for Sc5d. Since HMG CoA synthase is the first step in the cholesterol biosynthetic pathway, a decrease in brain cholesterol levels would be expected to increase the activity/expression of this enzyme, which is what was observed for both knockouts. In addition to changes in the level of protein expression, this approach also detected changes in protein post-translational modifications in brain proteins from knockout mice. These modifications were detected from a shift in protein mobility on the gel. An example of this change was observed for cofilin 1. Two gel spots were observed to contain cofilin 1, and the spot migrating with a more acidic isoelectric point was observed to have an increased intensity in knockout mice relative to wild type controls. Western Blots, using an anti-phospho cofilin 1 antibody, confirmed the increase in cofilin 1 phosphorylation. Another example of a shift in protein migration was observed for the Rab7 protein. Rab 7 is a member of a large family of GTP-binding proteins. Many of the proteins in this family are modified at the C-terminal by prenylation at C-terminal cysteine residues. Protein prenylation is a side branch in the biochemical pathway for cholesterol biosynthesis, and alternations in cholesterol biosynthesis could also result in changing the levels of protein prenylation. Prenylation is known to shift the mobility of at least some GTP-binding proteins, and the data suggest that a change in Rab7 prenylation may result in the observed mobility shift. However, a direct determination of Rab7 prenylation was not performed here, and other post-translational modifications may also be involved. 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. Quantification of Histone de-Methylation. Histone methylation regulates chromatin structure, transcription, and epigenetic state of the cell. Histone methylation is dynamically regulated by histone methylases and demethylases which mediate demethylation of di- and monomethylated histones. It has been unclear whether demethylases exist that reverse lysine trimethylation. A putative histone demethylase, KDM3B that was identified in earlier investigations, is being investigated to probe the specificity and extent of its demethylation capability using a model peptide synthesized with a lysine residue containing 1, 2 or 3 methylations. The methylated peptide, ARTKQTARKSTGGKAPRKQLAGGK-biotin, is incubated with either immuno-precipitated (IP) or recombinant KDM3B and the ratio of de-methylated product to substrate is determined by MALDI/TOF. Replicate mass spectral measurements of the peptide substrate from control and enzyme experiments, both with and without the putative co-factor RanQ69L-GTP, are corrected for background levels and the fractional de-methylation calculated. Initial studies with recombinant KDM3B showed no activity, while the IP enzyme showed activity for de-methylation of 1-Me, removal of both methyl groups from 2-Me and no activity with the 3-Me substrate.