Electrospray tandem mass spectrometry of human haemoglobin and a number of β-chain aberrants

Abstract

The use of electrospray tandem mass spectrometry is demonstrated for the characterisation of the polypeptide chains of normal human haemoglobin and a number of β-chain variants. Rapid screening of aberrant haemoglobins is achieved by the analysis of diluted blood without need for pretreatment or sample work-up. Gas-phase sequencing of the intact polypeptide chains shows that almost half of the amino acid sequence may be realised by this approach. More comprehensive coverage of the sequence (in excess of 80%) is given by cyanogen bromide digestion of whole blood followed by gas-phase sequencing of the resulting peptide fragments. The simplicity of the cyanogen bromide peptide map, accounting for the entire α- and β-chain sequences, is a distinct advantage of the method. Limitations of characterising aberrant proteins in triple quadrupole instruments are discussed with respect to mass accuracy and the effect of fragment ion charge state on detecting amino acid substitutions.