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.)
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