We describe a chemical proteomics approach to profile the interaction of small molecules with hundreds of endogenously expressed protein kinases and purine-binding proteins. This subproteome is captured by immobilized nonselective kinase inhibitors (kinobeads), and the bound proteins are quantified in parallel by mass spectrometry using isobaric tags for relative and absolute quantification (iTRAQ). By measuring the competition with the affinity matrix, we assess the binding of drugs to their targets in cell lysates and in cells. By mapping drug-induced changes in the phosphorylation state of the captured proteome, we also analyze signaling pathways downstream of target kinases. Quantitative profiling of the drugs imatinib (Gleevec), dasatinib (Sprycel) and bosutinib in K562 cells confirms known targets including ABL and SRC family kinases and identifies the receptor tyrosine kinase DDR1 and the oxidoreductase NQO2 as novel targets of imatinib. The data suggest that our approach is a valuable tool for drug discovery.

Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors

Protein purification using immobilised triazine dyes.

Perinatal exposure of rats to Bisphenol A affects the fertility of male offspring.

Frontal affinity chromatography: theory for its application to studies on specific interactions of biomolecules.

The affinity concept in bioseparation: evolving paradigms and expanding range of applications.

1. The suitability of cellulose and Sepharose as supports for affinity chromatography of two groups of cofactor-linked enzymes, dehydrogenases and kinases, was examined. Sepharose was found to be superior. 2. The selective capacities of the columns were measured by frontal analysis and are discussed in relation to the nucleotide contents. 3. The effect of various concentrations of enzyme and of non-specific protein on the performance of the affinity columns, and the effects of equilibration time, flow rate, sample volume and dilution of the nucleotide were examined. 4. The effect of interposing polymethylene and polyglycine extension arms between the matrix backbone and the nucleotide was investigated for several cofactor-dependent enzymes. Maximum binding was observed with an extension arm 0.8–1nm long.

/pdf/some-parameters-relevant-to-affinity-chromatography-on-3bioqgxdna.pdf

Some parameters relevant to affinity chromatography on immobilized nucleotides.

The effect of column geometry and dynamics on the interaction of glycerokinase and lactate dehydrogenase with N6-(6-aminohexyl)-5′-AMP-Sepharose has been studied. The strength of the enzyme-ligand interaction and the capacity of the adsorbent were shown to be complex functions related to column geometry, ligand concentration and total amount of ligand. The bed geometry had a significant effect on the strength of the interaction at low ligand concentrations and a particularly pronounced effect on interacting systems of relatively low affinity. Enzyme retardation was dependent upon ligand concentration and was independent of the concentration of both the applied enzyme sample and inert protein. The enzyme-ligand interaction, under column conditions, was shown to be a time-dependent process and evidence is presented to show that both the resolution and strength of interaction can be enhanced by increasing the enzyme-ligand equilibration time. Under batchwise conditions the apparent capacity of the adsorbent was dependent on both the enzyme concentration and the equilibration time. No difference was detected in the capacity of N6-(6-aminohexyl)-5′-AMP-Sepharose for glycerokinase and lactate dehydrogenase under batchwise conditions.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1974.tb03274.x

Affinity chromatography on immobilised adenosine 5'-monophosphate. Some kinetic parameters involved in the binding of group-specific enzymes.

1. The general applicability of affinity chromatography to the purification of nicotinamide nucleotide-dependent dehydrogenases on immobilized cofactors is illustrated with several examples taken from crude systems. 2. Methods for overcoming the inevitable loss of selectivity experienced with these polymers are suggested. Effective use of the appropriate nucleotide, the second substrate and other interacting ligands can be made to selectively alter the chromatographic behaviour of the desired enzyme.

/pdf/the-purification-of-nicotinamide-nucleotide-dependent-5eg04i132v.pdf

The purification of nicotinamide nucleotide-dependent dehydrogenases on immobilized cofactors.

Some applications of insolubilised cofactors to the purification of pyridine nucleotide-dependent dehydrogenases

Group specific matrices for affinity chromatography have now received considerable attention (1–9). Some of these studies have provided amongst the best examples of this aspect of affinity chromatography (10). Amongst the group specific matrices and in particular amongst the immobilised cofactors, the nicotinamide nucleotides present a very wide choice of applications and of different enzyme systems to study. However, because of the chemical complexity of the nicotinamide nucleotides, severe restrictions are imposed on the potential methods available for their immobilisation. These nucleotides may be immobilised readily onto cellulose or Sephadex or agarose containing aminocaproic acid (1,11,12). The resultant polymers are almost certainly of undefined nature (13) apart from being contaminated with unreacted carboxyl groups. The former need not necessarily be a drawback; the polymer preparation described by Allen & Majeries (10) does not specify which carboxyl groups of B1 2 are utilised in the linkage to the agarose and this undefined matrix is capable of purification factors of thousands. This is not so with nucleotides where the purification factors rarely exceed fifty times (1,8).

C. R. Lowe

Papers

Some parameters relevant to affinity chromatography on immobilized nucleotides.

Affinity chromatography on immobilised adenosine 5'-monophosphate. Some kinetic parameters involved in the binding of group-specific enzymes.

The purification of nicotinamide nucleotide-dependent dehydrogenases on immobilized cofactors.

Some applications of insolubilised cofactors to the purification of pyridine nucleotide-dependent dehydrogenases

An Analysis of Affinity Chromatography Using Immobilised Alkyl Nucleotides