C.S. Ho

Department of Chemical Pathology, Prince of Wales Hospital, Hong Kong

Molecular weight determination is one of the important parameters for protein characterisation. Traditional methods of protein molecular weight determination are, for example, composition analysis, sedimentation equilibrium / velocity, gel-filtration chromatography and polyacrylamide gel electrophoresis. Most of these methods suffer from poor precision and accuracy.

Modern mass spectrometry technique can be used to replace traditional methods for protein molecular weight determination. In this seminar, the principle of mass spectrometry is briefly introduced: methods of ionisation, mass analysers and the detectors. The experience of working with the Micromass Quattro II - a tandem mass spectrometry system will be used to illustrate how tandem mass spectrometry can be used for characterisation of protein molecules. On the Quattro II, electrospray ionisation technique is used to impart multiple charges onto protein molecules. The charged protein molecules are analysed by quadrupole mass analysers. The mass/charge ratios of the molecules are measured by a photomultiplier detector. Raw data of the signals are then analysed by computer software. This system is able to measure intact protein with molecular weight close to 100,000 daltons with an accuracy of ± 0.01% and precision CV of better than 0.01%. Furthermore, when the analyser is interfaced with a high performance liquid chromatrographic system, amino acid sequencing on tryptic digested peptides can be performed. The results allow peptide mapping with established database for identification of proteins structure.

Haemoglobin is used to demonstrate the functionalities of Quattro II for measurement of glycated haemoglobins and the determination of haemoglobin variants.

Copyright 1997 Hong Kong Medical Technology Association .
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