Two Glutaraldehyde‐Immobilized Trypsin Preparations for Peptide Mapping by Capillary Zone Electrophoresis, Liquid Chromatography, and Mass Spectrometry

Migneault, Isabelle; Dartiguenave, Catherine; Vinh, Joëlle; Bertrand, Michel; Waldron, Karen C.

doi:10.1080/10826070801890413

Cited by 14 publications

(6 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To demonstrate that the technique elaborated here was also capable of immobilizing proteins in their bioactive form, trypsin, a proteolytic enzyme, was immobilized onto the PS‐AEMA‐(GA‐TEPA) 3 ‐GA surface. Trypsin is responsible for cleaving peptide bonds during digestion, and is specifically active on arginine and lysine segments 46, 47. This specificity of trypsin can be used to determine its activity on various substrates 46, 48–52.…”

Section: Resultsmentioning

confidence: 99%

Surface‐functionalization of plasma‐treated polystyrene by hyperbranched polymers and use in biological applications

Boulares-Pender

Prager-Duschke

Elsner

et al. 2009

J of Applied Polymer Sci

View full text Add to dashboard Cite

Nitrogen plasma was used to amino-functionalize polystyrene surfaces, which were further modified via the selective introduction of polyamines suitable for the immobilization of biological compounds. This chemical modification was carried out using a multifunctional amine compound linked to glutaraldehyde, leading to the formation of hyperbranched structures at the surface. Up to three generations of branched polymers at the polystyrene (PS) surface were created by successive addition of the functional compounds. Amine functions introduced at the surface were labeled with 2,3,4,5,6-pentafluorobenzaldehyde and analyzed by X-ray photoelectron spectroscopy (XPS), confirming the successful attachment of each generation of branching. Finally, bovine serum albumin and trypsin were immobilized on N 2 -plasma-treated PS modified with different amounts of branched graft polymer and found to remain bioactive after immobilization.

show abstract

Section: Resultsmentioning

confidence: 99%

Surface‐functionalization of plasma‐treated polystyrene by hyperbranched polymers and use in biological applications

Boulares-Pender

Prager-Duschke

Elsner

et al. 2009

J of Applied Polymer Sci

View full text Add to dashboard Cite

show abstract

“…The following procedure was adapted from our previous method for trypsin and from preliminary studies using chymotrypsin . A 50 mM sodium phosphate buffer, pH 6.4, was used for several preparation and washing steps in this section.…”

Section: Methodsmentioning

confidence: 99%

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

2013

Self Cite

View full text Add to dashboard Cite

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.

show abstract

“…CZE with UV‐detection at 200 nm in sodium phosphate, pH 2.5, has been employed in the evaluation of two glutaraldehyde‐immobilized trypsin preparations, (i) covalent coupling to aminopropyl controlled pore glass particles and (ii) carrier‐free immobilization via glutaraldehyde crosslinking, for peptide mapping of lysozyme 240. Among the two preparations, the latter digested lysozyme more completely, but both of them represent reliable and catalytically active enzyme preparations that can be repeatedly used for faster proteolysis of dissolved protein substrates than solution‐phase and in‐gel proteolysis.…”

Section: Applicationsmentioning

confidence: 99%

Recent advances in CE and CEC of peptides (2007–2009)

Kašička

2009

Electrophoresis

View full text Add to dashboard Cite

The review presents a comprehensive survey of recent developments of high-performance capillary electromigration methods, zone electrophoresis, ITP, IEF, affinity electrophoresis, EKC and electrochromatography, and their application to analysis, preparation and physicochemical characterization of peptides. New approaches to the theoretical description and experimental investigation of electromigration properties of peptides, and to methodology of their separations, such as sample preparation, adsorption suppression, EOF control and detection, are described. New developments in particular CE and CEC modes are reported and several types of their applications to peptide analysis are presented: conventional qualitative and quantitative analysis, determination in biomatrices, monitoring of chemical and enzymatical reactions and physical changes, amino acid and sequence analysis, and peptide mapping of proteins. Few examples of micropreparative peptide separations are shown and capabilities of CE and CEC techniques to provide important physicochemical characteristics of peptides are demonstrated.

show abstract

Two Glutaraldehyde‐Immobilized Trypsin Preparations for Peptide Mapping by Capillary Zone Electrophoresis, Liquid Chromatography, and Mass Spectrometry

Cited by 14 publications

References 31 publications

Surface‐functionalization of plasma‐treated polystyrene by hyperbranched polymers and use in biological applications

Surface‐functionalization of plasma‐treated polystyrene by hyperbranched polymers and use in biological applications

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

Recent advances in CE and CEC of peptides (2007–2009)

Contact Info

Product

Resources

About