AUTOMATED ID OF PROTEINS, VARIANTS & POST-TRANSLATIONAL MODIFICATIONS

蛋白质、变体的自动识别

• 批准号: 7722961
• 负责人: DAVID H K CHUI
• 金额: $ 0.78万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7722961
• 关键词: Automation; Clinical; Computer Retrieval of Information on Scientific Projects Database; Coupled; Custom; DNA analysis; Data; Data Analyses; Databases; Detection; Development; Disease; Ensure; Freezing; Funding; Genes; Grant; Hemoglobin; Human; Institution; Laboratories; Measurement; Methodology; Methods; Modification; Operative Surgical Procedures; Peptides; Play; Population; Post-Translational Protein Processing; Preparation; Procedures; Process; Proteins; Proteomics; Protocols documentation; Research; Research Personnel; Research Project Grants; Resolution; Resources; Role; Sampling; Sickle Cell; Source; Standards of Weights and Measures; SwissProt; Techniques; Time; Trypsin; United States National Institutes of Health; Variant; Water; Whole Blood; base; computerized data processing; helix-loop-helix protein differentiation inhibitor; instrument; liquid chromatography mass spectrometry; programs; skills; success

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. More than 1200 recognized hemoglobin (Hb) structural variants exist in the human population, many of which may clinically manifest themselves as a disease state. Additionally, post-translational modifications (PTMs) of Hb that are not observed in gene-based analysis may play an important role in this disease. A reliable LC-MS/MS technique coupled with a proteomics based data analysis approach is needed to fully identify Hb variants and their PTMs. In this study, a robust and reliable LC-MS/MS proteomics based method for automated characterization of Hb protein variants and PTMs is being explored. Whole blood is washed with PBS, diluted and frozen prior to use. Proteins are separated and digested with trypsin and the digests are analyzed by online LC-MS/MS (QTOF-API-US, Waters Corporation). Accurate mass is calculated in real time by operation of a reference Lockspray. Data are processed using ProteinLynx Global Server 2.05 (Waters) and searched against SwissProt and custom programmed Hemoglobin/PTM databases. In our method, as minimal purification, derivatization or separation is required, the sample preparation is considerably simplified. Full automation of data analysis methodology has been established by which the biases and skill of an operator do not limit the success of the identification process. Data analysis time is reduced by using a pre-programmed Hb database search in which Hb-derived tryptic peptides (including variants and modifications) are unambiguously identified, while the peptides not derived from Hb can be digitally filtered out, based on the accuracies of mass measurement of the high resolution QTOF instrument. Various PTMs have also been programmed for automatic PTM search. This simplified sample preparation procedure and the automated data analysis are critical components of this proteomic approach in enabling high through-put sample analysis/data interpretation and ensure precise and accurate data consistency. Samples of Hb that cannot be analyzed by the standard CE-based protocols of the BUSM Sickle Cell laboratory are now frequently analyzed by LC/MS and MS/MS. LC-MS/MS allows the detection of variants, provides a rapid alternative to DNA analysis with additional information obtained for PTMs and has potential for clinical use. (A separately-described Core Research project is exploring the development of even more advanced approaches to this analytical challenge.)

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 人口中存在1200多个公认的血红蛋白（HB）结构变异，其中许多在临床上可能表现为疾病状态。此外，基于基因的分析中未观察到的HB的翻译后修饰（PTM）可能在该疾病中起重要作用。需要可靠的LC-MS/MS技术，再加上基于蛋白质组学的数据分析方法，以充分识别HB变体及其PTM。在这项研究中，正在探索一种基于强大而可靠的LC-MS/MS蛋白质组学方法，用于探索HB蛋白质变体和PTM的自动表征。全血被PBS洗涤，在使用前稀释和冷冻。蛋白质分离并用胰蛋白酶消化，并通过在线LC-MS/MS（QTOF-API-US，Waters Corporation）分析消化。准确的质量是通过参考锁定实时计算的。使用Proteinlynx Global Server 2.05（Waters）处理数据，并针对SwissProt和自定义编程的血红蛋白/PTM数据库进行搜索。在我们的方法中，由于需要最小的纯化，衍生化或分离，样品制备被大大简化。已经建立了数据分析方法的完全自动化方法，通过该方法，操作员的偏见和技能不会限制识别过程的成功。通过使用预编程的HB数据库搜索来减少数据分析时间，在该搜索中，基于高分辨率QTOF仪器的质量测量值的准确性，可以明确鉴定出HB衍生的胰蛋白酶肽（包括变体和修饰），而未从HB得出的肽可以被数字过滤掉。各种PTM也已被编程用于自动PTM搜索。这种简化的样本准备程序和自动数据分析是这种蛋白质组学方法的关键组成部分，可以实现高贯穿示例分析/数据解释并确保精确，准确的数据一致性。现在经常通过LC/MS和MS/MS分析HB不能通过BUSM Sickle Cell实验室的标准CE基准方案进行分析的HB样本。 LC-MS/MS允许检测变体，提供了DNA分析的快速替代方法，并提供了用于PTM的其他信息，并且具有临床使用的潜力。 （单独描述的核心研究项目正在探索更高级的方法来应对这种分析挑战。）