Electrospray mass spectrometry in the clinical diagnosis of variant hemoglobins.
A combination of mass spectrometric (MS) techniques [electrospray MS, liquid secondary ion MS (LSIMS) and MS-MS] has been used for variant hemoglobin (Hb) detection and characterization. Electrospray MS allowed analysis of mixtures of intact globins giving simultaneously the molecular weights (accuracy 1-2 Da) and information about relative amounts of globins present. Currently, 14 Da is the minimum molecular weight difference required experimentally to accurately measure different species present in a mixture of 15-16 kDa proteins. Thus 80 and 79% of the known variants of alpha and beta chains, respectively, can be detected in mixtures with their normal counterparts, including Hb S (molecular weight difference = 30 Da). Abnormal hemoglobins detected were fractionated by C4 reversed-phase high-performance liquid chromatography (HPLC), and the separated globin chains (or the mixture of whole precipitated globin) were digested by trypsin. The tryptic peptides were separated by C18 reversed-phase HPLC and analyzed by LSIMS to narrow down the mutation site to a single peptide. The mass measured in LSIMS frequently corresponded to a unique structure, thus giving the unequivocal identification of the mutation and its site. Where there was ambiguity, tandem MS on a Kratos Concept four-sector instrument was used for sequencing the abnormal peptide. The practical use of the methodologies presented is illustrated through analysis and identification of Hb G-San Jose, Hb Stanleyville II, Hb S and Hb Willamette.
Shackleton
C H
CH
Clinical Mass Spectrometry Facility, Children's Hospital Research Institute, Oakland, CA 94609.
Falick
A M
AM
Green
B N
BN
Witkowska
H E
HE
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D005914:Globins / Q000032:analysis / Q000737:chemistry; D006454:Hemoglobins / Q000032:analysis; D006455:Hemoglobins, Abnormal / Q000032:analysis*; D006801:Humans; D007525:Isoelectric Focusing; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014357:Trypsin
D006454:Hemoglobins; D006455:Hemoglobins, Abnormal; C037389:hemoglobin Stanleyville II; C034872:hemoglobin Willamette; D005914:Globins; C032126:hemoglobin G San Jose; D014357:Trypsin