Synthesis and Evaluation of a Novel Adenosine-Ribose Probe for Global-Scale Profiling of Nucleoside and Nucleotide-Binding Proteins

Abstract

Proteomics is a powerful approach used for investigating the complex molecular mechanisms of disease pathogenesis and progression. An important challenge in modern protein profiling approaches involves targeting of specific protein activities in order to identify altered molecular processes associated with disease pathophysiology. Adenosine-binding proteins represent an important subset of the proteome where aberrant expression or activity changes of these proteins have been implicated in numerous human diseases. Herein, we describe an affinity-based approach for the enrichment of adenosine-binding proteins from a complex cell proteome. A novel N 6-biotinylated-8-azido-adenosine probe (AdoR probe) was synthesized, which contains a reactive group that forms a covalent bond with the target proteins, as well as a biotin tag for affinity enrichment using avidin chromatography. Probe specificity was confirmed with protein standards prior to further evaluation in a complex protein mixture consisting of a lysate derived from mouse neuroblastoma N18TG2 cells. Protein identification and relative quantitation using mass spectrometry allowed for the identification of small variations in abundance of nucleoside- and nucleotide-binding proteins in these samples where a significant enrichment of AdoR-binding proteins in the labeled proteome from the neuroblastoma cells was observed. The results from this study demonstrate the utility of this method to enrich for nucleoside- and nucleotide-binding proteins in a complex protein mixture, pointing towards a unique set of proteins that can be examined in the context of further understanding mechanisms of disease, or fundamental biological processes in general.

Binding-based proteomic profiling and the fatty acid amides

“…BBPP probe-labeled proteins can be visualized and/or purified by either incorporating a biotin or fluorescent group directly into the probe or incorporating an alkyne moiety into the probe to “click-in” [34] a biotin or fluorescent group (R*) into the probe after the probe-labeled proteins have been produced. We have designed BBPP probes based on 8-azido-adenosine analogues to profile adenosine/adenylated binding proteins in mouse neuroblastoma N 18 TG 2 cells [35] and Niphakis et al . [36] generated diazirine-containing analogs of anandamide to profile anandamide-binding proteins in HEK293T cells.…”

A Photo-clickable ATP-Mimetic Reveals Nucleotide Interactors in the Membrane Proteome

“…So far, different nucleotide resins and functionalized ATP probes have been used to identify ATP interactors from complex proteomes (Hanoulle et al, 2006;Ito et al, 2006;Mahajan et al, 2015;Patricelli et al, 2007). As previously discussed (Mahajan et al, 2015), site, size, and properties of the functional group(s) attached to ATP biases the interaction profile of the respective probe. Meaningful ATP interaction profiles can only be expected if the probe design does not interfere with ATP's allosteric or enzymatic functions of interest.…”

Adenosine: Synthetic Methods of Its Derivatives and Antitumor Activity

“…Under this same synthetic line, some products were identified being nucleotides obtained as adenosine derivatives and which can act as transcriptional primers. These derivatives corresponded to a series of amino adenosine compounds, which were synthesized by a method assisted from 5'-aminophosphoramidate and water-soluble carbodiimide, generating a direct coupling of diamines with AMP [57]. Fundamentally, chlorophosphorylation of 6-chloropurine riboside was carried out, followed by hydrolysis of 6-chloropurine riboside-5-monophosphate and a displacement or substitution of 6-chloride diamines later, obtaining N 6 -adenosine derivatives.…”

“…Subsequently, two series of adenosine nucleotides were synthesized which were coupled and conjugated to groups of fluorescein and biotin at the extreme 5’ or at the N 6 position, through linkers of different sizes and hydrophobicities. Both adenosine nucleotides series could act as efficient transcription initiators, producing fluorescein-labeled and biotin-labeled RNA at the extreme 5'-end under a “ one-step ” procedure, eliminating post-transcriptional modification [57].…”