Mass spectrometry to detect the site specificity of advanced glycation/lipoxidation end-product formation on protein: some challenges and solutions

Formation of AGEs (advanced glycation end-products) and ALEs (advanced lipoxidation end-products) on proteins is associated with aging and various diseases of oxidative stress, notably diabetes and its complications. Modification of protein to AGE/ALEs is known to be site-directed and this has potential implications for protein functionality and design of AGE/ALE inhibitors. Determination of the site-specificity of modification is achieved most efficiently by MS. The present paper summarizes some of the challenges that need to be addressed when determining the site-specificity of AGE/ALE formation on protein by MS, using the protein RNase as an example. The following topics are discussed: formation and significance of AGE/ALEs, location of glycated peptides, enzymic digestion of glycated peptides and selection of mass spectrometric settings of analysis for glycated peptides.