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

From hundreds to thousands: Widening the normal human Urinome (1)

“…The phase deriving from butanol extraction was lyophilized and loaded onto a column of 150 mL peptide library beads equilibrated in 25 mM phosphate buffer, pH 7.4 as already described by Candiano et al [17,18]. The eluate and the unbound fraction were preserved at À 80 1C until analysis by mass spectrometry.…”

Detailed Methodologies and Protocols

“…Nevertheless, if amino acids are associated with agents that weaken most important interactions such as hydrophobic (e.g., with ethylene glycol), ionic (e.g., with sodium of potassium chloride), and hydrogen bonding (e.g., with urea), the probability of protein elution happening is high. It is with this in mind that the first attempts to elute using this general rule were successfully made for urinary proteins by Candiano et al (2012).…”

Mark Twain: How to fathom the depth of your pet proteome

“…those trace proteins that had to compete hard with the overwhelming presence of the high-abundance species (HAP) that might have had lower affinities for the same baits! As a result of the insufficient elution protocol, only the high-to medium-abundance species were desorbed, [14] 4 M urea, 1% CHAPS Only high abundance proteins 1 μg to 40 mg/mL Bandhakavi et al [50] Candiano et al [24] 100 mM Tris-HCl pH 6 [14]. This series of experiments also demonstrated the importance of ionic strength upon protein capture.…”

“…Although we have just stated that the best eluant from CPLLs is boiling 4% SDS in the presence of 30 mM DTT, we have chanced upon a novel, mild elution system, taking place under physiological conditions [24], which seems to go against the grain of what has been preached above. It consists in challenging the beads with a mixture of Lys-Arg-Asp-Glu (D E R K, dubbed the Fab Four, at 50, 50, 25, and 25 mM final concentrations, respectively, of each of the four).…”