AUTOMATED ID OF PROTEINS, VARIANTS & POST-TRANSLATIONAL MODIFICATIONS

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 人类中存在超过1200种公认的血红蛋白（Hb）结构变体，其中许多可能在临床上表现为疾病状态。此外，在基于基因的分析中未观察到的Hb的翻译后修饰（PTM）可能在这种疾病中起重要作用。需要一种可靠的LC-MS/MS技术与基于蛋白质组学的数据分析方法相结合，以充分鉴定Hb变体及其PTM。在这项研究中，一个强大的和可靠的LC-MS/MS蛋白质组学为基础的方法，Hb蛋白质变体和PTM的自动化表征正在探索。使用前，用PBS洗涤全血，稀释并冷冻。分离蛋白质并用胰蛋白酶消化，并通过在线LC-MS/MS（QTOF-API-US，沃茨Corporation）分析胰蛋白酶。通过操作参考Lockspray，真实的实时计算精确质量。使用ProteinLynx Global Server 2.05（沃茨）处理数据，并针对SwissProt和定制编程的血红蛋白/PTM数据库进行检索。在我们的方法中，由于需要最少的纯化、衍生化或分离，样品制备大大简化。数据分析方法的完全自动化已经建立，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，其中Hb衍生的胰蛋白酶肽（包括变体和修饰）被明确地鉴定，而不是来自Hb的肽可以基于高分辨率QTOF仪器的质量测量的准确性被数字地过滤掉。各种PTM也被编程为自动PTM搜索。这种简化的样品制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，可以实现高通量样品分析/数据解释，并确保精确和准确的数据一致性。不能通过BUSM镰状细胞实验室基于CE的标准方案进行分析的Hb样品现在经常通过LC/MS和MS/MS进行分析。LC-MS/MS允许检测变体，提供了DNA分析的快速替代方法，并获得了PTM的额外信息，具有临床应用潜力。（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.)