Characterization Of Proteins By Mass Spectrometry

Smith-Lemli-Opitz综合征（SLOS）的脑蛋白质组学。本研究使用小鼠模型研究涉及胆固醇生物合成缺陷的人类遗传疾病。对野生型和敲除小鼠（Dhcr7和Sc5d）脑组织进行二维凝胶蛋白质组学研究，通过对三次重复二维凝胶的图像分析，确定了64种表达差异超过1.5倍的蛋白质。对于蛋白质鉴定，至少有3个肽段的匹配置信度大于95%，氨基酸序列覆盖范围为总蛋白质的14%至74%。HMG辅酶a合成酶是脑提取物中观察到的差异表达蛋白的一个例子。敲除动物脑HMG CoA合成酶显著高于野生型，Dhcr7与敲除动物脑HMG CoA合成酶的比值为1:1.55，Sc5d为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，脑胆固醇水平的降低将有望增加该酶的活性/表达，这是在两个基因敲除中观察到的。除了蛋白表达水平的变化外，该方法还检测了敲除小鼠脑蛋白翻译后修饰的变化。这些修饰是通过凝胶上蛋白质流动性的变化来检测的。在cofilin 1中观察到这种变化的一个例子。两个凝胶点被观察到含有cofilin 1，并且与野生型对照相比，在敲除小鼠中观察到与更酸的等电点迁移的斑点强度增加。Western Blots使用抗磷酸化cofilin 1抗体，证实cofilin 1磷酸化增加。另一个蛋白质迁移转移的例子是Rab7蛋白。Rab 7是gtp结合蛋白大家族的一员。这个家族中的许多蛋白质在c端通过c端半胱氨酸残基的戊烯化修饰。蛋白质烯酰化是胆固醇生物合成生化途径中的一个侧分支，胆固醇生物合成的改变也可能导致蛋白质烯酰化水平的改变。已知戊酰化可以改变至少一些gtp结合蛋白的迁移性，数据表明Rab7戊酰化的改变可能导致观察到的迁移性变化。然而，这里没有直接测定Rab7戊烯酰化，也可能涉及其他翻译后修饰。

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.