Analysis of human serum albumin variants by mass spectrometric procedures

A new strategy for the structural characterisation of human albumin variants has been developed which makes extensive use of mass spectrometric methodologies. The rationale behind the method is to provide a rapid and effective screening of the entire albumin structure. The first step in this strategy consists in the attempt to determine the accurate molecular mass of the intact variant by electrospray mass spectrometry often providing a first indication on the presence of the variant. An HPLC procedure has been developed io isolate all the seven fragments generated by CNBr hydrolysis of HSA in a single chromatographic step. A rapid screening of the entire albumin structure is achieved by the ESMS analysis of the peptide fragments and the protein region(s) carrying the structural abnormality is identified by its anomalous mass value(s). Mass mapping of the corresponding CNBr peptide, either by Fast Atom Bombardment Mass Spectrometry (FABMS) or by Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDIMS), leads to the definition of the site and the nature of the variation. This combined strategy was applied to the structural characterisation of three HSA genetic variants and provided to be an effective procedure for the rapid assessment of their structural modifications showing considerable advantages over the classical approach.