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.