Characterization Of Proteins and Other Molecules By Mass Spectrometry

通过质谱法表征蛋白质和其他分子

• 批准号: 8351268
• 负责人: ALFRED L YERGEY
• 金额: $ 93.96万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 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/8351268
• 关键词: Adult; Age; Alzheimer' s Disease; Antibiotic Therapy; Area; Biological; Biological Assay; Biological Markers; Brain; Cardiolipins; Cell Wall; Cerebrospinal Fluid; Cessation of life; Charge; Childhood; Clinical Research; Code; Collection; Complex; Consult; Coupled; Data; Databases; Disease; Disulfides; Experimental Designs; Female; Fingerprint; Gel; Half-Life; Human; Individual; Infant; Injection of therapeutic agent; Institutes; Ions; Lipids; Liposomes; Liquid substance; Mass Spectrum Analysis; Measurement; Medical center; Membrane; Methodology; Methods; Miglustat; Modeling; Molecular Weight; Monitor; Mothers; Mus; National Institute of Child Health and Human Development; Nerve Degeneration; Newborn Infant; Nuclear Pore Complex; Organism; Patients; Penicillins; Peptides; Phase; Phosphorylation; Post-Translational Protein Processing; Prealbumin; Pregnant Women; Principal Component Analysis; Probability; Process; Proteins; Proteomics; Quality Control; Recombinant Proteins; Recovery; Research Personnel; Respiratory distress; Role; Sampling; Saturated Fatty Acids; Serum; Severity of illness; Sheep; Shotguns; Site; Solid; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spottings; Steroids; Supraoptic Vertical Ophthalmoplegia; System; Techniques; Time; Variant; Workload; base; candidate identification; experience; falls; fatty acid-binding proteins; instrument; instrumentation; interest; mutant; novel; novel strategies; protein aminoacid sequence; protein profiling; research study; sample collection; skills; sugar; synthetic protein

Protein Characterization in Niemann-Pick Disease-Type C (NPC). Differentially expressed protein spots (R&#8805;1.20, p<0.05 and R&#8804;0.83, p<0.05) were isolated from Npc1 mutant female mice and littermate controls at 1, 3 and 5 weeks of age, and using mass spectrometric techniques, we have candidate identifications to date on more than 200 pairs of protein spots. Functional assays of a number of candidates with potential involvement with liposomal storage diseases and neuronal degeneration is continuing on the proteins identified. To date two candidates of particular interest are transthyretin and fatty acid binding protein. Although these protein identifications were made from mouse brain, both of these are intriguing potential biomarkers in human CSF. One reason for this is that decreased levels of cerebral spinal fluid transthyretin correlate with disease severity in Alzheimer disease and elevated levels of fatty acid binding protein have been determined in CSF of NPC patients. 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 using MALDI TOF (Matrix Assisted Laser Desorption Ionization-Time Of Flight) mass spectrometry. 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. In addition to the use of our Analysis Of VAriance coupled with Principal Component Analysis (ANOVA-PCA) analytical approach to MALDI-TOF profiling, we have begun the process of confirming proteins found in these same CSF samples through the use of shotgun proteomics methodology, albeit on less complex systems than those to which this approach is generally applied. The proteins in the CSF will be quantified once digested for shotgun analysis using isotopically coded tags, iTRAQ. Lipid Quantification in Serum. We have continued developing methodology to quantify cardiolipins in human serum. 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 200 uL 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. Recent measurements using a new instrument (sensitivity more than 100-fold greater and with mass accuracy <10 ppm for the singly charged ions) have shown consistently that (18:2)-4 levels in normal adult serum vary between 3 and 10 fmole/uL, with biological variations far greater than replicate measurements of an individual sample. In addition, other cardiolipins with saturated fatty acid moieties have been detected in several individuals. New Approach to Mass Spectrometric-Based Protein Identification. Current approaches to protein identification rely heavily on database matching of fragmentation spectra, while ignoring (in a scoring sense) the mass accuracy of the precursor ions. We have developed a method based originally upon MALDI TOF-TOF instrumentation that uses targeted peptide mass fingerprinting results to confirm MS/MS database search identifications. The method uses first-order spectral data that have heretofore been ignored by most search engines. In this method the distribution of mass errors of peptide matches in the first order spectrum is used to develop a probability model that is independent from MS/MS database search identifications. Peptide mass matches can come from both precursor ions that have been fragmented as well as those that are tentatively identified by accurate mass alone. This additional confirmation enables us to assign protein identifications to MS/MS based scores that are otherwise considered to be only of moderate quality. The probability model employed uses Bayesian Inference and the mass errors in the first-order spectra to assign peptide probabilities and the product of individual peptide probabilities yields the final protein scores. Results based on a commercially available standard mixture of intact proteins shows both very high confidence levels for the proteins found in a 1D gel separation of the mixture, but also has also demonstrated that so-called one hit wonders found in fragmentation experiments can be validated reliably using this method. We suggest that the method will be applicable to LC-MS/MS methodologies that have precursor mass ion accuracies of at least 100 ppm.

C 型尼曼匹克病 (NPC) 的蛋白质表征。从 1、3 和 5 周龄的 Npc1 突变雌性小鼠和同窝对照小鼠中分离出差异表达的蛋白点（R≥1.20，p<0.05 和 R≤0.83，p<0.05），并使用质谱技术，迄今为止我们对 200 多对蛋白点进行了候选鉴定。对一些可能与脂质体贮积病和神经元变性有关的候选蛋白的功能测定仍在继续进行。迄今为止，两个特别令人感兴趣的候选物是运甲状腺素蛋白和脂肪酸结合蛋白。尽管这些蛋白质鉴定是从小鼠大脑中进行的，但这两种蛋白质都是人类脑脊液中令人感兴趣的潜在生物标志物。其原因之一是脑脊液运甲状腺素蛋白水平降低与阿尔茨海默病的疾病严重程度相关，并且已在鼻咽癌患者的脑脊液中确定了脂肪酸结合蛋白水平升高。 NPC 的脑脊液分析 (CSF)。初步实验表明，我们可以使用 MALDI TOF（基质辅助激光解吸电离飞行时间）质谱法比较蛋白质谱的新颖分析方法，检测鼻咽癌患者和成人对照的脑脊液之间的显着差异。从 37 名特征明确的鼻咽癌患者身上采集了脑脊液样本。目前，鼻咽癌患者的收集包括 2.5 年多时间收集的纵向样本和 5 对 miglustat 前/后样本。此外，国家儿童医疗中心正在收集儿科脑脊液样本。除了使用我们的方差分析与主成分分析 (ANOVA-PCA) 分析方法进行 MALDI-TOF 分析外，我们还开始通过使用鸟枪法蛋白质组学方法来确认这些相同 CSF 样本中发现的蛋白质，尽管系统比通常应用该方法的系统复杂度要低。脑脊液中的蛋白质在消化后将使用同位素编码标签 iTRAQ 进行鸟枪法分析。 血清中的脂质定量。我们继续开发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已证明，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，这种物质在血清中注射约 100 pmole/mL 的水平会导致呼吸窘迫；请注意，该浓度迅速下降，半衰期只有几分钟。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，通过液-液和固相组合进行提取，然后进行 LC-MS 分析，其中包含提取/回收标准品以监测系统质量控制。我们已经证明，可以以大约 90% 的效率从血清中提取心磷脂。我们进一步表明，正常成人血清中 (18:2)-4 心磷脂的水平 < 10 fmole/uL，比早期不太准确的测量结果低约 1000 倍。最近使用新仪器进行的测量（灵敏度提高了 100 倍以上，单电荷离子的质量精度 <10 ppm）一致表明，正常成人血清中的 (18:2)-4 水平在 3 至 10 fmole/uL 之间变化，其生物变化远远大于单个样品的重复测量。此外，在几个个体中还检测到了其他具有饱和脂肪酸部分的心磷脂。 基于质谱的蛋白质鉴定的新方法。当前的蛋白质鉴定方法严重依赖于碎片谱的数据库匹配，而忽略（在评分意义上）前体离子的质量准确性。 我们开发了一种最初基于 MALDI TOF-TOF 仪器的方法，该方法使用目标肽质量指纹图谱结果来确认 MS/MS 数据库搜索识别。 该方法使用迄今为止被大多数搜索引擎忽略的一阶光谱数据。 在此方法中，使用一阶谱中肽匹配的质量误差分布来开发独立于 MS/MS 数据库搜索识别的概率模型。 肽质量匹配可以来自已裂解的前体离子以及仅通过精确质量初步鉴定的前体离子。 这一额外的确认使我们能够将蛋白质鉴定分配给基于 MS/MS 的分数，否则这些分数被认为仅具有中等质量。所采用的概率模型使用贝叶斯推理和一阶光谱中的质量误差来分配肽概率，并且单个肽概率的乘积产生最终的蛋白质分数。基于市售的完整蛋白质标准混合物的结果显示，在混合物的一维凝胶分离中发现的蛋白质的置信度非常高，而且还证明了使用这种方法可以可靠地验证在片段化实验中发现的所谓“一击奇迹”。我们建议该方法适用于前体质量离子精度至少为 100 ppm 的 LC-MS/MS 方法。