Widening and Diversifying the Proteome Capture by Combinatorial Peptide Ligand Libraries via Alcian Blue Dye Binding

Abstract

Combinatorial peptide ligand libraries (CPLLs) tend to bind complex molecules such as dyes due to their aromatic, heterocyclic, hydrophobic, and ionic nature that may affect the protein capture specificity. In this experimental work Alcian Blue 8GX, a positively charged phthalocyanine dye well-known to bind to glycoproteins and to glucosaminoglycans, was adsorbed on a chemically modified CPLL solid phase, and the behavior of the resulting conjugate was then investigated. The control and dye-adsorbed beads were used to harvest the human urinary proteome at physiological pH, this resulting in a grand total of 1151 gene products identified after the capture. Although the Alcian Blue-modified CPLL incremented the total protein capture by 115 species, it particularly enriched some families among the harvested proteins, such as glycoproteins and nucleotide-binding proteins. This study teaches that it is possible, via the two combined harvest mechanisms, to drive the CPLL capture toward the enrichment of specific protein categories.

Current state of the art for enhancing urine biomarker discovery

“…The effectiveness of these fractionation strategies can be manipulated by varying the physicochemical properties of the system. Changes in salinity, protein concentration, ionic strength, and/or pH can alter protein-protein interactions by a) modifying adsorption to solid phase columns, b) altering the strength of hexapeptide-protein interactions, or c) by enhancing or inhibiting hydrophobic interactions between affinity baits and proteins, or between hexapeptides and complementary proteins [6, 25, 104-107]. For example, increased salt concentration can denature antibodies thus inhibiting solid phase affinity capture [105].…”

“…For example, increased salt concentration can denature antibodies thus inhibiting solid phase affinity capture [105]. Decreasing the ionic strength, increasing hydrophobic interactions, and maintaining solutions within pH 4-9 of CPLL solutions can improve protein enrichment/depletion strategies using combinatorial peptide ligand libraries (discussed further below) [6, 107]. …”

“…The newest iteration of CPLL contain Alcian blue dye coupled to succinylated hexamers of hexapeptides to enhance protein binding [6]. By adding the copper containing phthalocyanine dye, Alcian blue, to the succinylated hexamers of CPLL, 115 urinary proteins with nucleotide, carbohydrate or cation binding, or catalytic activity were detected [6].…”

“…By adding the copper containing phthalocyanine dye, Alcian blue, to the succinylated hexamers of CPLL, 115 urinary proteins with nucleotide, carbohydrate or cation binding, or catalytic activity were detected [6]. Combining CPLL equilibration techniques and physicochemical dye interactions into one capture moiety has revealed 38 previously undetected urine proteins, increasing the total urinary proteome to 4430 gene products [6]. By substituting the dye or altering the functional group of the hexapeptide libraries via different chemical transformations, a plethora of new protein harvesting agents can be synthesized to explore the urinary proteome.…”

“…For example, manual annotation of the literature by the UniProt consortium has revealed between 13,841 and 17,132 human protein-coding genes [5]. Yet, by 2015, only 4,430 urinary proteins had been identified, a large number of which were detected due to recent improvements in pre-analytical fractionation/concentration techniques [6, 7]. …”