AUTOMATED ID OF PROTEINS, VARIANTS & POST-TRANSLATIONAL MODIFICATIONS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。人类中存在1200多种已知的血红蛋白（Hb）结构变异，其中许多可能在临床上表现为一种疾病状态。此外，在基于基因的分析中未观察到的Hb的翻译后修饰（PTMs）可能在该疾病中发挥重要作用。需要可靠的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变体及其PTMs。在这项研究中，一种基于LC-MS/MS的蛋白质组学方法正在探索Hb蛋白变异和PTMs的自动表征。全血在使用前用PBS洗涤，稀释并冷冻。蛋白质用胰蛋白酶分离消化，用在线LC-MS/MS （QTOF-API-US, Waters Corporation）分析。通过参考锁喷器的操作实时计算出准确的质量。数据使用ProteinLynx Global Server 2.05 （Waters）进行处理，并与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，由于只需要很少的纯化、衍生或分离，因此样品制备相当简化。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。通过使用预先编程的Hb数据库搜索减少数据分析时间，其中Hb衍生的色氨酸肽（包括变体和修饰）被明确识别，而基于高分辨率QTOF仪器的质量测量精度，非Hb衍生的肽可以被数字过滤掉。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的标准方案分析的Hb样品现在经常使用LC/MS和MS/MS进行分析。LC-MS/MS允许检测变异，为PTMs提供快速替代DNA分析的附加信息，具有临床应用的潜力。（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.)