AUTOMATED ID OF PROTEINS, VARIANTS & POST-TRANSLATIONAL MODIFICATIONS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 人类群体中存在着1200多种已知的血红蛋白(Hb)结构变体，其中许多在临床上可能表现为疾病状态。此外，在基于基因的分析中没有观察到的Hb的翻译后修饰(PTM)可能在这种疾病中起着重要作用。需要一种可靠的LC-MS/MS技术结合基于蛋白质组学的数据分析方法来充分识别Hb变异体及其PTM。在这项研究中，一种基于LC-MS/MS蛋白质组学的稳健和可靠的方法正在探索用于Hb蛋白变体和PTMS的自动化表征。全血在使用前用PBS洗涤、稀释和冷冻。蛋白质用胰酶分离和消化，用在线LC-MS/MS(QTOF-API-US，Waters Corporation)分析。通过参考锁喷雾的操作实时计算出准确的质量。使用ProteinLynx Global Server 2.05(Waters)处理数据，并根据SwissProt和定制编程的血红蛋白/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，因此样品的制备相当简单。已经建立了数据分析方法的完全自动化，通过这种方法，操作员的偏见和技能不会限制识别过程的成功。通过使用预先编程的Hb数据库搜索来缩短数据分析时间，其中，基于高分辨率QTOF仪器的质量测量精度，可以明确地识别Hb衍生的胰多肽(包括变体和修饰)，同时可以数字过滤出非Hb衍生的多肽。还对各种PTM进行了编程，以便自动进行PTM搜索。这种简化的样品准备程序和自动化数据分析是这种蛋白质组学方法的关键组成部分，有助于实现高通量样品分析/数据解释，并确保准确和准确的数据一致性。不能通过BUSM Sickle Cell实验室基于CE的标准协议分析的Hb样本现在经常通过LC/MS和MS/MS进行分析，LC-MS/MS可以检测变异，为DNA分析提供了一种快速替代DNA分析的方法，为PTM获得了更多信息，具有临床应用的潜力。(另一个单独描述的核心研究项目正在探索开发更先进的方法来应对这一分析挑战。)

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.)