EXPEDITING RARE VARIANT HEMOGLOBIN CHARACTERIZATION BY COMBINED HPLC ELECTROSPRAY MASS-SPECTROMETRY

Microscale analysis of a variant hemoglobin (Hb) has been achieved by combination of high performance liquid chromatography (HPLC) and elect rospray, mass spectrometry (ESMS) and the method should be almost univ ersally applicable. We have eliminated preparative scale HPLC of globi n chains and semi-preparative HPLC of proteolytic digests which had be en used prior to mass spectrometry. Use of microbore HPLC columns redu ced the time required for analysis substantially and solvent usage by 100x. Molecular masses of intact globins and masses and sequence infor mation of tryptic peptides could be obtained without collecting and se parately analyzing chromatographic fractions. As an example of the use of these methods, we report the characterization of an unknown hemogl obinopathy case that was finally authenticated as Hb P-Galveston{beta1 17(G19)His->Arg], using the following sequence of analyses: 1) ESMS of complete hemolysate, 2) analytical HPLC of globin chains, 3) combined microbore HPLC/ESMS of globin chains to determine their molecular mas ses, 4) cysteine derivatization and tryptic digestion of mixture of al l globins, followed by microbore separation of the peptides, molecular mass determination, and generation of fragmentation patterns allowing confirmation of amino acid sequences. This four-pan strategy should a llow characterization of almost all variant Hbs. Exceptions would be m utations in regions of globin chains which give rise to small (< four residues) tryptic peptides, either normal or produced by addition of n ew tryptic sites and mutations that introduce only minute difference i n molecular weight (MW) of tryptic peptides. Since only 10% of each se parated peptides is mass analyzed, 90% is available for collection and further structural identification (e.g. by tandem MS or Edman sequenc ing) if the identity is still in doubt.