“Cheek-to-cheek” urinary proteome profiling via combinatorial peptide ligand libraries: A novel, unexpected elution system

Abstract

A new method is here reported for facile elution of the human urinary proteome after being captured with combinatorial peptide ligand libraries (CPLL, ProteoMiner). It consists in challenging the beads with 100mM Tris, pH 7.4, or with 100mM Lys, pH 7.4 or even better with a mixture of Lys, Arg, Asp and Glu (150mM final concentration). These elutions permit recovery of species in a native form, for monitoring any biological activity of the eluted species, while avoiding the noxious presence of sodium dodecyl sulphate (SDS), reported as the best eluant so far from CPLL beads. SDS, albeit permitting quantitative recovery from the beads, has to be removed from the sample prior to mass spectrometry analysis. This unorthodox elution, which most likely will work only for urine samples, seems to be due to the fact that bile salts and urinary pigments are massively adsorbed by the beads, thus masking the hydrophobic binding sites of aromatic and non-aromatic amino acids. The binding thus occurs mostly via ionic and hydrogen bond interactions via the "Grand Catchers" Arg, Lys, His, which can then be easily challenged by positively charged species, such a Tris, free Lys and free Arg in the eluant as well as by negatively charged compounds, such as Glu and Asp. When eluting with the four-amino acid mix, at least 3300 spots can be visualized in a 2D map.