Application of immobilized metal affinity chromatography in proteomics

It has been proved that the progress of proteomics is mostly determined by the development of advanced and sensitive protein separation technologies. Immobilized metal affinity chromatography (IMAC) is a powerful protein fractionation method used to enrich metal-associated proteins and peptides. In proteomics, IMAC has been widely employed as a prefractionation method to increase the resolution in protein separation. The combination of IMAC with other protein analytical technologies has been successfully utilized to characterize metalloproteome and post-translational modifications. In the near future, newly developed IMAC integrated with other proteomic methods will greatly contribute to the revolution of expression, cell-mapping and structural proteomics.     

Application of immobilized metal affinity chromatography in proteomics

It has been proved that the progress of proteomics is mostly determined by the development of advanced and sensitive protein separation technologies. Immobilized metal affinity chromatography (IMAC) is a powerful protein fractionation method used to enrich metal-associated proteins and peptides. In proteomics, IMAC has been widely employed as a prefractionation method to increase the resolution in protein separation. The combination of IMAC with other protein analytical technologies has been successfully utilized to characterize metalloproteome and post-translational modifications. In the near future, newly developed IMAC integrated with other proteomic methods will greatly contribute to the revolution of expression, cell-mapping and structural proteomics.     

Chemical proteomics for drug discovery based on compound-immobilized affinity chromatography

Chemical proteomics is an effective approach to focused proteomics, having the potential to find specific interactors in moderate-scale comprehensive analysis. Unlike chemical genetics, chemical proteomics directly and comprehensively identifies proteins that bind specifically to candidate compounds by means of affinity chromatographic purification using the immobilized candidate, combined with mass spectrometric identification of interacting proteins. This is an effective approach for discovering unknown protein functions, identifying the molecular mechanisms of drug action, and obtaining information for optimization of lead compounds. However, immobilized-small molecule affinity chromatography always suffers from the problem of non-specific binders. Although several approaches were reported to reduce non-specific binding proteins, these are mainly focused on the use of low-binding-affinity beads or insertion of a spacer between the bead and the compound. Stable isotope labeling strategies have proven particularly advantageous for the discrimination of true interactors from many non-specific binders, including carrier proteins, such as serum albumin, and are expected to be valuable for drug discovery.     

Evaluating immobilized metal affinity chromatography for the selection of histidine-containing peptides in comparative proteomics

Agarose based immobilized metal affinity chromatography (IMAC) columns loaded with copper (II) were evaluated for the selection of histidine-containing peptides in comparative proteomics. Recovery, binding specificity, and reproducibility were investigated with model proteins. Cu(II)-IMAC was found to be highly selective for histidine containing peptides; moreover, a low degree of nonspecific selection was observed. Acylation of the amino-terminus of peptides with either succinic anhydride, N-acetoxysuccinamide, or [3-(2,5)-dioxopyrrolidin-1-yloxycarbonyl)-propyl]-trimethylammonium (quaternary amine) reduced the number of histidine-containing peptides bound by the Cu(II)-IMAC columns. This provides an additional possibility for sample simplification in proteomic applications. The number of acylated peptides selected decreased in the order of quaternary amine > N-acetoxysuccinamide > succinic anhydride derivatization. Although the selection of N-terminally derivatized peptides is biased toward peptides that contain more than one histidine, it is not yet possible to predict selectivity.     

Applications of affinity chromatography to the study of drug-melanin binding interactions

This short review reports on progresses in the study of drug-melanin interactions using the technique of affinity chromatography. Melanins are natural or synthetic pigments derived from the oxidation and polymerization of various precursors including L-dopa, tyrosine and cystein. Accumulation of toxic compounds, drugs, and metal ions in pigmented tissues through reversible binding to melanin has been linked to chronic toxicity. Affinity chromatography using chromatographic stationary phases based on physically adsorbed or chemically bonded melanin provides a useful tool for studying the interactions of small molecules and metal ions with melanin     

Erythrocyte stroma included in polyacrylamide gel. Applications to affinity chromatography]

Stromata prepared with human or animal red blood cells are suspended in acrylamide solution. Gel as prepared for electrophoresis is dispersed, before use, and can be used in chromatographic columns for the retention of agglutinins. Lectins, e.g., where first absorbed and then eluted with solution of inhibitory sugar or with acid buffer. Some applications are mentioned.     

O-linked N-acetylglucosamine proteomics of postsynaptic density preparations using lectin weak affinity chromatography and mass spectrometry

O-GlcNAc is a widespread dynamic carbohydrate modification of cytosolic and nuclear proteins with features analogous to phosphorylation. O-GlcNAc acts critically in many cellular processes, including signal transduction, protein degradation, and regulation of gene expression. However, the study of its specific regulatory functions has been limited by difficulties in mapping sites of O-GlcNAc modification. We report methods for direct enrichment and identification of in vivo O-GlcNAc-modified peptides through lectin weak affinity chromatography (LWAC) and mass spectrometry. The effectiveness of this strategy on complex peptide mixtures was demonstrated through enrichment of 145 unique O-GlcNAc-modified peptides from a postsynaptic density preparation. 65 of these O-GlcNAc-modified peptides were sequenced and belonged to proteins with diverse functions in synaptic transmission. Beta-elimination/Michael addition, MS(3) on O-GlcNAc neutral loss ions, and electron capture dissociation were shown to facilitate analysis of O-GlcNAc-modified peptides/sites from lectin weak affinity chromatography enriched postsynaptic density samples. Bassoon and Piccolo, proteins critical to synapse assembly and vesicle docking, were extensively modified by O-GlcNAc. In some cases, O-GlcNAc was mapped to peptides previously identified as phosphorylated, indicating potential interplay between these modifications. Shared substrate amino acid context was apparent in subsets of O-GlcNAc-modified peptides, including "PVST" and a novel "TTA" motif (two hydroxyl-containing amino acids adjacent to an alanine). The results suggest specific roles for O-GlcNAc modification in synaptic transmission, establish a basis for site-specific regulatory studies, and provide methods that will facilitate O-GlcNAc proteome analysis across a wide variety of cells and tissues.     

Scaling-up affinity chromatography

Recently in Trends in Biotechnology (Luong, J. T. et al., Vol. 5, 281–286), it was argued that affinity chromatography had disadvantages in productivity and operation and that other methods of exploiting affinity interactions needed to be developed. It has taken a long time for affinity methods to become accepted in biological processing, and it will take longer for new techniques to make a significant impact. In the meantime, affinity chromatography is being scaled up.     

Lectin affinity chromatography

A protocol is described for the preparation of lectin affinity chromatography columns using purified lectins and preactivated matrices. A general method is given for the purification of glycoproteins on immobilized Con A. Methods for immobilizing Con A on CDI agarose, Affi-Gel 15, and carbonyl-diimidazole-activated agarose are described.     

Applications of proteomics in hepatic diseases research

Proteomics has become an important part in the leading research area and been widely used in the disease-associated study. In hepatic research field, proteomics could be applied in study of hepatic diseases including liver cancer, cirrhosis and hepatotoxicities, etc. Significant proteins could be identified as biomarkers, drug targets and clues for pathogenesis illumination.     

Applications of proteomics in hepatic diseases research

Since the completion of human genome draft in the process of Human Genome Project (HGP), life science has entered the post-genome era in the early 21st century. Proteomics has become an important part in the leading research area and been applied in many fields of life science such as the occurrence and de-velopment of tumor, cell differentiation and embryo-genesis, mechanisms of gene regulation, mechanisms and therapy of major diseases, pharmacy, relationship between environment and health …     

Applications of urinary proteomics in biomarker discovery

Urine is an important source of biomarkers. This article reviews current advances, major challenges, and future prospects in the field of urinary proteomics. Because the practical clinical problem is to distinguish diseases with similar symptoms, merely comparing samples from patients of a particular disease to those of healthy individuals is inadequate for finding biomarkers with sufficient diagnostic power. In addition, the variation of expression levels of urinary proteins among healthy individuals and individuals under different physiological conditions adds to the difficulty in identifying biomarkers. We propose that establishing the natural variation in urinary protein expression among a healthy population can serve as a reference to help identify protein abundance changes that are caused by disease, not by individual variations or physiological changes. We also discuss that comparing protein expression levels between urine and plasma may reveal the physiological function of the kidney and that may facilitate biomarker discovery. Finally, we propose that establishing a data-sharing platform for data collection and integrating results from all urinary biomarker studies will help promote the development of urinary proteomics.     

Aptamers as affinity reagents for clinical proteomics

Application of proteomic results to scientific and medical practice will depend in many respects on progress of affinity microchips technologies. This determines continuous search for inexpensive and robust affinity reagents alternative to monoclonal antibodies. Among synthetic mimetics of antibodies, the oligonucleotide aptamers are of the greatest interest as the affinity reagents due to the possibility to automate their selection and due to the low cost of oligonucleotide synthesis. In the review we consider the problems related to the automation and optimization of aptamer selection and also to selection of photoaptamers capable to form photoinduced covalent complexes with the protein targets. The existing approaches to the post-selection modification of the aptamers to increase their affinity and selectivity to protein targets are discussed.     

New developments in affinity chromatography

The design, synthesis and chromatographic operation of a new range of stable and selective immobilized dye affinity adsorbents for potential application in the purification of pharmaceutical proteins is described. Computer aided molecular design has been exploited to design novel dye ligands which show a predictable selectivity for the target protein and which, when coupled to stable perfluoropolymer supports, yield high capacity, low leakage adsorbents for affinity chromatography. It is anticipated that these new materials will withstand the rigorous conditions required for sanitization and cleaning in situ of industrial scale processes.     

Semicontinuous affinity chromatography separations

The semicontinuous operation of affinity chromatography columns charged and eluted alternately is studied mathematically. A strongly nonlinear adsorption equilibrium relation is utilized to examine the periodic mode of adsorption and desorption. The governing differential equations are solved by the method of characteristics. The time interval between desorption and adsorption periods is estimated, and implications for separations are discussed to show the advantages of semicontinuous cyclic operation.     

Quantitative affinity chromatography.

The objective of this review is to summarize developments in the use of quantitative affinity chromatography to determine equilibrium constants for solute interactions of biological interest. Affinity chromatography is an extremely versatile method for characterizing interactions between dissimilar reactants because the biospecificity incorporated into the design of the affinity matrix ensures applicability of the method regardless of the relative sizes of the two reacting solutes. Adoption of different experimental strategies, such as column chromatography, simple partition equilibrium experiments, solid-phase immunoassay, and biosensor technology, has led to a situation whereby affinity chromatography affords a means of characterizing interactions governed by an extremely broad range of binding affinities--relatively weak interactions (binding constants below 10(3) M(-1)) through to interactions with binding constants in excess of 10(9) M(-1). In addition to its important role in solute separation and purification, affinity chromatography thus also possesses considerable potential for investigating the functional roles of the reactants thereby purified.