Advanced sorbents for preparative protein separation purposes

Boschetti, Egisto

doi:10.1016/0021-9673(94)80017-0

Cited by 180 publications

(94 citation statements)

References 61 publications

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“…A solution of protein molecular weight markers was used to further assess the molecular weight cut off (MWCO) of the proteins sequestered by the particles ( Figure 8B 39 . In order to further indagate molecular sieving properties of the beads, NIPAm/AAc particles were incubated with platelet derived growth factor B (0.003 mg/mL, 14,500 Da, Cell Signaling) and BSA (0.067 mg/mL) in Tris (100 mM pH 7) for one hour.…”

Section: Nih-pa Author Manuscriptmentioning

confidence: 99%

Smart Hydrogel Particles: Biomarker Harvesting: One-Step Affinity Purification, Size Exclusion, and Protection against Degradation

Luchini

Geho²,

Bishop³

et al. 2007

Nano Lett.

177

249

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Disease-associated blood biomarkers exist in exceedingly low concentrations within complex mixtures of high-abundance proteins such as albumin. We have introduced an affinity bait molecule into N-isopropylacrylamide to produce a particle that will perform three independent functions within minutes, in one step, in solution: a) molecular size sieving b) affinity capture of all solution phase target molecules, and c) complete protection of harvested proteins from enzymatic degradation. The captured analytes can be readily electroeluted for analysis.There is an urgent need to discover novel biomarkers that provide sensitive and specific disease detection1 , 2. Cancer is rapidly becoming the leading cause of death for many population groups in the United States, largely due to the fact that the disease is usually diagnosed after the cancer has metastasized and treatment is ineffective. It is widely believed that early detection of cancer prior to metastasis will lead to a dramatic improvement in treatment outcome. Biomarkers are nucleic acids, proteins, protein fragments or metabolites indicative of a specific biological state, that are associated with the risk of contraction or presence of disease3. Biomarker research has revealed that low-abundance circulating proteins and peptides present a rich source of information regarding the state of the organism as a whole 4 . Two major hurdles have prevented these discoveries from reaching clinical benefit: 1) disease-relevant biomarkers in blood or body fluids may exist in exceedingly low concentrations within a complex mixture of biomolecules and could be masked by high-abundance species such as albumin, and 2) degradation of protein biomarkers can occur immediately following the collection of blood or body fluid as a result of endogenous or exogenous proteinases. The goal of this study was to create "smart" nano-particles that allow enrichment and encapsulation of selected classes of proteins and peptides from complex mixtures of biomolecules such as plasma, and protect them from degradation during subsequent sample handling. The captured analytes can be readily extracted from the particles by electrophoresis allowing for subsequent quantitative analysis. This nanotechnology provides a powerful tool that is uniquely suited for the discovery of novel biomarkers for early stage diseases such as cancer.SUPPORTING INFORMATION AVAILABLE: Available in the Supplementary Information are details on particles synthesis protocol, SDS PAGE analysis on molecular sieving properties and enzymatic degradation, and tables (Table S1 and S2) listing proteins (with peptide coverage lists) identified via LC-MS/MS (ESI) on material electroeluted from NIPAm and NIPAm/AAc particles. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public AccessAuthor Manuscript Nano Lett. Author manuscript; available in PMC 2010 May 28. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe concentration of proteins and peptides comprising t...

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Section: Nih-pa Author Manuscriptmentioning

confidence: 99%

Smart Hydrogel Particles: Biomarker Harvesting: One-Step Affinity Purification, Size Exclusion, and Protection against Degradation

Luchini

Geho²,

Bishop³

et al. 2007

Nano Lett.

177

249

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“…Ion exchange chromatography, introduced for protein separations in the 1950s, remains one of the most widely used preparative-scale purification and separation methods (1). Ion exchange adsorbents have been designed to offer a range of mechanical and chemical properties for chromatographic separation and purification (2), but a fundamental understanding of transport mechanisms in these materials remains elusive. For most materials, simple diffusive mechanisms are thought to control intraparticle transport, but studies using batch or column techniques (3-7) yield model-dependent transport coefficients, and they cannot definitively determine the physical transport mechanism.…”

mentioning

confidence: 99%

Nondiffusive mechanisms enhance protein uptake rates in ion exchange particles

Dziennik

Belcher²,

Barker³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

127

145

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Scanning confocal fluorescence microscopy and multiphoton fluorescence microscopy were used to image the uptake of the protein lysozyme into individual ion exchange chromatography particles in a packed bed in real time. Self-sharpening concentration fronts penetrating into the particles were observed at low salt concentrations in all of the adsorbents studied, but persisted to 100 mM ionic strength only in some materials. In other adsorbents, diffuse profiles were seen at these higher salt concentrations, with the transition region exhibiting a pronounced fluorescence peak at the front at intermediate salt concentrations. These patterns in the uptake profiles are accompanied by significant increases in protein uptake rates that are also seen macroscopically in batch uptake experiments. The fluorescence peak appears to be a concentration overshoot that may develop, in part, from an electrokinetic contribution to transport that also enhances the uptake rate. Further evidence for an electrokinetic origin is that the effect is correlated with high adsorbent surface charge densities. Predictions of a mathematical model incorporating the electrokinetic effect are in qualitative agreement with the observations. These findings indicate that mechanisms other than diffusion contribute to protein transport in oppositely charged porous materials and may be exploited to achieve rapid uptake in process chromatography.T he dramatic growth of the biotechnology industry in recent years has depended critically on preparative separation techniques for protein products. Ion exchange chromatography, introduced for protein separations in the 1950s, remains one of the most widely used preparative-scale purification and separation methods (1). Ion exchange adsorbents have been designed to offer a range of mechanical and chemical properties for chromatographic separation and purification (2), but a fundamental understanding of transport mechanisms in these materials remains elusive. For most materials, simple diffusive mechanisms are thought to control intraparticle transport, but studies using batch or column techniques (3-7) yield model-dependent transport coefficients, and they cannot definitively determine the physical transport mechanism. With the projected acceleration in the discovery and development of protein therapeutics as a result of the human genome project, the demand for improved chromatographic materials and methods for optimizing protein separation and purification will increase. Therefore, a more fundamental understanding of the transport mechanisms in these materials is required to develop better heuristics for process development as well as methodologies for the design of new adsorbents.A key complication in the interpretation of batch or column studies is that the transport is coupled to adsorption, making additional assumptions regarding the adsorption behavior necessary to characterize the transport. Microscopic techniques have a greater potential to resolve the resulting ambiguities regarding transport mechanisms...

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“…An extensive list of the literature can be found in the reviews by Boschetti [72] and Narayanan [73], where tables of sorbents, their properties and commercial products can also be found. In general, classical soft gels based on dextran, diluted polyacrylamide and non-cross-linked agarose have gradually been replaced by more rigid materials, leading to improvements in the speed of separations through the use of higher mobile-phase flow rates, in the efficiency through the use of smaller and more regular stationary phase particles and in the resolution through the use of special separation mechanisms, such as affinity and hydrophobic interactions.…”

Section: Supports and Their Modificationmentioning

confidence: 99%

“…Newly synthesized Dalargin analogues, nonapeptides from intrachinary regions A6-14 of insulin, IGF I and IGF II, have been separated both by RPC and CE; CE is preferable for peptides available in extremely small quantities [96]. For preparative chromatography, see the reviews by Boschetti [72] and Narayanan [73] (Section 4.1). Micropreparative HPLC of peptides and proteins has also been reviewed [97].…”

Section: Reversed-phase Chromatographymentioning

confidence: 99%

Stationary phases for peptide analysis by high performance liquid chromatography: a review

Štulı́k¹,

Pacáková²,

Suchánková³

et al. 1997

Analytica Chimica Acta

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A survey is given of modern stationary phases employed in high performance liquid chromatography (HPLC) analysis of peptides. The physico-chemical properties of peptides and their consequences for the selection and optimization of the separation system are briefly discussed, followed by a summary of the approaches to the selection and characterization of stationary phases. The properties and applicability of various stationary phases are then critically reviewed, including aspects such as size-exclusion, ion-exchange, reversed-phase, hydrophobic-interaction, affinity and chiral systems, as well as some specialized separation techniques. Emphasis is placed on the most recent literature. 0 1997 Elsevier Science B.V.

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Advanced sorbents for preparative protein separation purposes

Cited by 180 publications

References 61 publications

Smart Hydrogel Particles: Biomarker Harvesting: One-Step Affinity Purification, Size Exclusion, and Protection against Degradation

Smart Hydrogel Particles: Biomarker Harvesting: One-Step Affinity Purification, Size Exclusion, and Protection against Degradation

Nondiffusive mechanisms enhance protein uptake rates in ion exchange particles

Stationary phases for peptide analysis by high performance liquid chromatography: a review

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