• atomic fluorescence
• mercury
• proteins

Chemical labelling in combination with mass spectrometry is appointed as a modern approach for quantifying biopolymers, especially proteins. Protein labelling approaches are generally based on elemental mass spectrometry techniques, specifically inductively coupled plasma-mass spectrometry (ICP-MS).
In this work we present a novel method for the characterization and determination of proteins labelled with p-hydroxymercurybenzoate (pHMB, an organic mercury species widely used for mercaptan and thiolic compound labelling), based on the on line oxidation of pHMB-labelled proteins with a novel on-line UV/microwave (MW) photochemical reactor, followed by cold vapour generation atomic fluorescence spectrometry (CVG-AFS) detection. MW/UV process leaded to the quantitative conversion of pHMB and protein-pHMB bioconjugates to Hg(II), with a yield of 89±0.5% without using chemical oxidating reagents and avoiding the use of toxic carcinogenic compounds.
LC-MW/UV-CVGAFS method has been applied to the characterization, separation and determination of several thiolic proteins (albumins, beta-lactoglobulin and k-casein) using SEC and RPC. Several denaturation systems (8 M urea, 3 M GdnSCN, 6 M GdnHCl, 0.2% SDS, 20% methanol, 50% trifluoroethanol) have been employed to study pHMB-ovalbumin bioconjugates, using ovalbumin as a model protein. The maximum number of titrated SH- groups for OVA has been obtained denaturing the protein with 0.2% SDS at room temperature (2.7 ±0.3 SH- groups). The kinetics of the binding of pHMB with OVA was also investigated.
SEC-MW/UV-CVGAFS was also been applied to the study of thiolic proteins in raw matrices such as human plasma, human saliva and milk, in order to demonstrate the analytical performance of this technique in the study of real samples. Due to the well assessed role of thiolic proteins in physio-pathology this methodology could allow a preliminary screening to identify unique markers of oxidative stress.
The potentialities of RPC-MW/UV-CVGAFS were explored for the study thiolic fragments in triptic digests and the effect of ageing on OVA in aqueous extracts of fresh and aged OVA-cinnabar paint replicas. The aim of this part of the work was to show how RPC-MW/UV-CVG-AFS system coupled to pHMB labeling i) could be applied to identify CYS containing peptides, ii) could be employed to identify species-specific proteins from the chromatographic elution pattern.