Immobilization of trypsin onto 1,4-diisothiocyanatobenzene-activated porous glass for microreactor-based peptide mapping by capillary electrophoresis: Effect of calcium ions on the immobilization procedure

Dartiguenave, Catherine; Hamad, Hussein; Waldron, Karen C.

doi:10.1016/j.aca.2010.01.042

Cited by 23 publications

(17 citation statements)

References 35 publications

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“…Except for the immobilization strategies, a suitable carrier is the key point for immobilization of trypsin, especially when connected with practical applications. Various carriers have been prepared such as inorganic porous carriers (10,11,19,20), natural polymeric carriers (21,22), magnetic carriers (9,23,24), and synthesis polymeric carriers (14,25) etc., for investigating the immobilization of trypsin. For example, Lee et al (9) have introduced magnetic nano-particles for the immobilization of trypsin to establish a rapid and efficient protein digestion.…”

Section: Introductionmentioning

confidence: 99%

Chitosan Modified PSt-GMA Microspheres With/Without Spacer-Arms as Carriers: Their Influences on Kinetics, Stability, Optimal pH, Adsorption Behavior of Immobilized Trypsin

Peng

Zhao

Liu

et al. 2012

Journal of Macromolecular Science, Part A

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A series of microspheres, chitosan modified PSt-GMA microspheres with/without the spacer-arms, have been used as carriers to study related properties about immobilized trypsin, such as isothermal adsorption, stability, kinetics and optimal reaction pH. The experimental results showed that the immobilization process could fit the Freundlich model rather than the Langmuir model, indicating the real adsorption behavior was more complex than the ideal adsorption model. In addition, K m values of trypsin immobilized onto M5 or M8 were closer to that of free enzyme and lower than that onto other microspheres, suggesting the trypsin immobilized onto M5 or M8 possessed a higher affinity to substrate. Furthermore, the thermal and operational stabilities of immobilized trypsin, particularly onto M5 or M8, were enhanced compared to that of free trypsin. Compared to free trypsin, the optimal pH of immobilized trypsin didn't change; however,its pH profile was broader. These results have proved that chitosan modified microspheres could keep the conformation and activity of enzyme as well as enhance enzyme's properties. Therefore, it has potential application in high throughput screening of drugs (HTS) based on the scintillation proximity assay (SPA) method.

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Section: Introductionmentioning

confidence: 99%

Chitosan Modified PSt-GMA Microspheres With/Without Spacer-Arms as Carriers: Their Influences on Kinetics, Stability, Optimal pH, Adsorption Behavior of Immobilized Trypsin

Peng

Zhao

Liu

et al. 2012

Journal of Macromolecular Science, Part A

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“…Immobilization is proposed to decrease autoproteolysis by reducing the enzyme's flexibility . Trypsin immobilized via the ε‐amino groups of lysine residues is inherently stabilized against autoproteolysis because a large proportion of its cleavage sites are literally “tied up” . Chymotrypsin, which cleaves substrate at aromatic residues, does not benefit from this, so it is important to carefully evaluate the autoproteolysis of this enzyme once it is insolubilized.…”

Section: Resultsmentioning

confidence: 99%

Development of glutaraldehyde‐crosslinked chymotrypsin and an in situ immobilized enzyme microreactor with peptide mapping by capillary electrophoresis

2013

Self Cite

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Immobilized proteolytic enzymes present several advantages over their soluble form, not the least of which is suppression of autoproteolysis peaks even at high enzyme-to-substrate ratios. We have made immobilized chymotrypsin by directly crosslinking it with glutaraldehyde to produce polymeric particles. Digestion of two model substrates using the particles was followed by CE peptide mapping with detection by UV absorbance or LIF. Results showed that autoproteolysis was highly suppressed and that different storage conditions of the particles in the short term (24 h) did not affect digestion of denatured BSA. As well, the chymotrypsin particles were indifferent to the presence of fluorescein groups on a casein substrate. Glutaraldehyde crosslinking of chymotrypsin inside a fused silica capillary column to make an immobilized enzyme reactor (IMER) was achieved in a series of reagent addition and washing steps, entirely automated using a commercial CE instrument. Digestion of myoglobin in the IMER for 30 min at 37°C followed by peptide mapping by CE-MS of the collected digest allowed identification of 17 chymotryptic peptides of myoglobin, or 83% primary sequence coverage.

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“…Peptide maps of β‐casein obtained by CZE with UV‐detection at 200 nm in 50 mM sodium phosphate BGE, pH 2.5, were used for evaluation of immobilization conditions and kinetic behavior of trypsin covalently immobilized via the 1,4‐diisothiocyanatobenzene linker onto aminopropyl controlled pore glass particles 251. The immobilized trypsin could be repeatedly used for fast (few min) hydrolysis of proteins and subsequent peptide mapping.…”

Section: Applicationsmentioning

confidence: 99%

Recent developments in CE and CEC of peptides (2009–2011)

Kašička

2011

Electrophoresis

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The review brings a comprehensive survey of the recent developments of high-performance electroseparation methods in capillary and microchip formats: zone electrophoresis, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography and electrochromatography. Applications of these techniques to analysis, isolation, purification and physicochemical and biochemical characterization of peptides are described. Advances in the investigation of electromigration properties of peptides, and in the methodology of their analysis, such as sample preparation, adsorption suppression, EOF control and detection, are presented. New developments, in particular, CE and CEC modes are reported and several types of their applications to peptide analysis are described: conventional qualitative and quantitative analysis, determination in complex (bio)matrices, monitoring of chemical and enzymatical reactions and physical changes, amino acid, sequence and chiral analysis, and peptide mapping of proteins. Some micropreparative peptide separations are shown and capabilities of CE and CEC techniques to provide relevant physicochemical characteristics of peptides are demonstrated.

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Immobilization of trypsin onto 1,4-diisothiocyanatobenzene-activated porous glass for microreactor-based peptide mapping by capillary electrophoresis: Effect of calcium ions on the immobilization procedure

Cited by 23 publications

References 35 publications

Chitosan Modified PSt-GMA Microspheres With/Without Spacer-Arms as Carriers: Their Influences on Kinetics, Stability, Optimal pH, Adsorption Behavior of Immobilized Trypsin

Chitosan Modified PSt-GMA Microspheres With/Without Spacer-Arms as Carriers: Their Influences on Kinetics, Stability, Optimal pH, Adsorption Behavior of Immobilized Trypsin

Development of glutaraldehyde‐crosslinked chymotrypsin and an in situ immobilized enzyme microreactor with peptide mapping by capillary electrophoresis

Recent developments in CE and CEC of peptides (2009–2011)

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