Proteinaceous binders identification in the works of art using ion-pairing free reversed-phase liquid chromatography coupled with tandem mass spectrometry

Abstract

A simple, fast and reliable procedure for the proteinaceous binders identification in the works of art samples is presented. The procedure consisted of ammonia extraction in order to suppress pigment interferences, acidic hydrolysis and quantification of underivatized amino acids using reversed phase liquid chromatography coupled with electrospray tandem mass spectrometry (RP-LC–ESI-MS/MS). Fourteen underivatized amino acids were quantified without the addition of ion-pairing agents (IPA) using the multiple reaction monitoring (MRM) mode. The chromatographic separation was optimized by testing three C18 columns and three different eluent compositions. The optimal chromatographic resolution and the ionization efficiency were achieved with Symmetry C18 column and the eluent consisting of water with methanol. The amino acids composition of the proteins commonly found in the paint binding media, eggs, casein and animal glues, was determined. The procedure was tested using a set of naturally aged samples. Calculated detection and quantification limits indicated that the method is suitable for the analysis of protein binders in the paint micro-samples. Animal glues, casein and eggs were identified in the samples from 18^th and 19^th century paintings by Jacek Malczewski, while eggs and casein were detected in the mural painting samples from the 13^th century UNESCO-listed church of Yemrehanna Krestos. The LC/MS/MS based method of the protein binders identification described here can be used as an alternative for the approach based on the gas chromatography coupled with mass spectrometry (GC/MS).