Children's Hospital Oakland Research Institute (CHORI) is a center for diagnosis and treatment of hemoglobinopathies. Over 400 known genetic variants of human hemoglobin thathave been described so far and presumably many more as yet uncharacterized. Hemoglobin variants have amino acid substitutions in the protein chain, and arise as a consequence of single basesubstitutions within the globin coding gene loci. In HbS (sickle cell) for instance, a single basemutation changing A-->T in the triplet codon for glutamic acid results in the substitution b6Glu-->6Val. The severity of conditions produced by the clinically important variants, especially when present in a homozygous state (for example HbSS) or in a heterozygous mixture withnon-compatible counterparts justify the growing interest in variant hemoglobin detection and identification. Complete characterization of suspected variant hemoglobins detected by screening (by isoelectric focusing or ion-exchange HPLC) or from clinical symptoms begins with proteolytic enzyme cleavage (trypsin) followed by HPLC separation of the resulting peptide mixture. Initially, abnormal peptides were sequenced by Edman degradation, but mass spectrometry has now become the method of choice for analyzing the tryptic fragments. Recently, three mass spectrometric techniques, 4-sector tandem mass spectrometry, electrospray and MALDI TOF haveemerged as powerful new tools for peptide and protein characterization. Electrospray MS and MALDI MS allow analysis of mixtures of intact globins, giving simultaneously the molecular weights and information about relative amounts of globins present. These techniques alsohave the advantage that they are """"""""charge independent"""""""" methods and hve the potential to identify many variants not detectable by isoelectric focusing. Oncea variant peptide has been detected in a digest, tandem mass spectrometry provides a rapid and efficient method for complete characterization of its sequence. This technique is particularly advantageous in the case of hemoglobin peptides because the variants typically differ only slightly from the very well-known normal peptides. A 4-sector tandem instrument is the instrument of choice due to the superior parent and daughter ion resolution that is afforded by suchan instrument. However, the Fisons TOF SPEC SE MALDI instrument at the resource should also be able to accurately sequence many of the proteolytic fragments using the new post-source decay technique. The combination of mass spectrometric techniques (electrospray, MALDI TOF, LSIMS and MS/MS) that are available in the UCSF Mass Spectrometry Facility is ideally suited to complete characterization of mutant hemoglobins. Hemoglobin samples of interest will be made available fromscreening and clinical studies at Children's Hospital Oakland and will have previously been analyzed by standard electrospray techniques carried out at that institution, which is a major screening center for Northern California. It should be emphasized that screening of large numbers of hemoglobin samples will not be carried out at UCSF. Our interest lies primarily in studying unknown hemoglobins that require advanced techniques for identification and/or characterization.
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