ICP-MS and MALDI-TOF: an integrated approach in the study of metal-protein interactions in biological samples

The activity and stability of most of proteins in a biological organism are subject to metal ion binding. Heavy metals such as arsenic and mercury are able to interact with proteins and, due to their impact on human health and environmental pollution, they represent a priority investigation field. Moreover, the contribution of these metals to the pathological processes, requires an in-depth understanding of the protein targets. Studying the nature of these interactions require the most appropriate analytical method. In order to verify the most suitable operational conditions, the in vitro interactions in proteins-rich foodstuffs (such as: milk, eggs and food grade gelatin) with some As and Hg compounds have been evaluated. Then, some naturally exposed samples have been analyzed in order to study any in vivo interactions. Samples have been separated using SEC chromatography and metals eluted have been detected by ICP-MS, while proteins have been identified by MALDI-TOF. The results showed that arsenical compounds had strictly species-specific interactions, while for mercury compounds fewer differences have been found. Since the use of ICP-MS for the analysis of biomolecules containing heteroatoms can give us several advantages in terms of sensitivity and specificity, a part of this PhD thesis has been devoted to protein labeling techniques. The study has been focussed on the development of an HPLC-ICP-MS hetero-atom tagging method for the identification of intact proteins isolated from painting layers. The proteins labeling has been performed using the metal element-chelated tag (MECT) procedure. The analytical procedure has been firstly tested on proteins standard. Then, the characterization of proteinaceous binders typically used in paints from past centuries has been performed. The proteins extraction and purification procedures have been evaluated using reconstructed pictorial models before testing the effectiveness of the method on some unknown samples.