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C-terminal ladder sequencing of peptides using an alternative nucleophile in carboxypeptidase Y digests
KTH, School of Biotechnology (BIO), Biochemistry.
KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
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2006 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 357, no 2, 167-172 p.Article in journal (Refereed) Published
Abstract [en]

 A method for improved sequence coverage in C-terminal sequencing of peptides, based on carboxypeptidase digestion, is described. In conventional carboxypeptidase digestions, the peptide substrate is usually extensively degraded and a full amino acid sequence cannot be obtained due to the lack of a complete peptide ladder. In the presented method, a protecting group is introduced at the C terminus of a fraction of the peptide fragments formed in the digest, and thereby further degradation of the C-terminally modified peptides are slowed down. The protecting group was attached to the C-terminal amino acid through a carboxypeptidase-catalyzed reaction with an alternative nucleophile, 2-pyridylmethylamine, added to the aqueous digestion buffer. Six peptides were digested by carboxypeptidase Y with and without 2-pyridylmethylamine present in the digest buffer, and the resulting fragments subsequently were analyzed with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Comparison of the two digestion methods showed that the probability of successful ladder sequencing increased, by more than 50% using 2-pyridylmethylamine as a competing nucleophile in carboxypeptidase Y digests.

Place, publisher, year, edition, pages
2006. Vol. 357, no 2, 167-172 p.
Keyword [en]
2-Pyridylmethylamine; MALDI; Matrix-assisted laser desorption/ionization mass spectrometry; Transpeptidation
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-7106DOI: 10.1016/j.ab.2006.07.025ISI: 000241077800002Scopus ID: 2-s2.0-33748784378OAI: oai:DiVA.org:kth-7106DiVA: diva2:12019
Note
QC20100524Available from: 2007-05-15 Created: 2007-05-15 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Enzyme selectivity as a tool in analytical chemistry
Open this publication in new window or tab >>Enzyme selectivity as a tool in analytical chemistry
2007 (English)Licentiate thesis, comprehensive summary (Other scientific)
Abstract [en]

Enzymes are useful tools as specific analytical reagents. Two different analysis methods were developed for use in the separate fields of protein science and organic synthesis. Both methods rely on the substrate specificity of enzymes. Enzyme catalysis and substrate specificity is described and put in context with each of the two developed methods.

In paper I a method for C-terminal peptide sequencing was developed based on conventional Carboxypeptidase Y digestion combined with matrix assisted laser desorption/ionization mass spectrometry. An alternative nucleophile was used to obtain a stable peptide ladder and improve sequence coverage.

In paper II and III, three different enzymes were used for rapid analysis of enantiomeric excess and conversion of O-acylated cyanohydrins synthesized by a defined protocol. Horse liver alcohol dehydrogenase, Candida antarctica lipase B and pig liver esterase were sequentially added to a solution containing the O-acylated cyanohydrin. Each enzyme caused a drop in absorbance from oxidation of NADH to NAD+. The conversion and enantiomeric excess of the sample could be calculated from the relative differences in absorbance.

Place, publisher, year, edition, pages
Stockholm: KTH, 2007. viii, 35 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2007:5
Keyword
substrate specificity, competing substrates, enantiomeric excess analysis, library screening, ladder sequencing, carboxypeptidase Y, 2-pyridylmethylamine, MALDI, Candia antarctica lipase B, pig liver esterase, horse liver alcohol dehydrogenase, acylated cyanohydrins
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-4377 (URN)978-91-7178-674-6 (ISBN)
Presentation
2007-06-01, FB54, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20101108Available from: 2007-05-15 Created: 2007-05-15 Last updated: 2010-11-08Bibliographically approved
2. Serine Hydrolase Selectivity: Kinetics and applications in organic and analytical chemistry
Open this publication in new window or tab >>Serine Hydrolase Selectivity: Kinetics and applications in organic and analytical chemistry
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The substrate selectivities for different serine hydrolases were utilized in various applications, presented in papers I-VI. The articles are discussed in the thesis in view of the kinetics of the enzyme catalysis involved.

In paper I the enantioselectivities towards a range of secondary alcohols were reversed for Candida antarctica lipase B by site directed mutagenesis. The thermodynamic components of the enantioselectivity were determined for the mutated variant of the lipase.

In papers II-III Candida antarctica lipase B was engineered for selective monoacylation using two different approaches. A variant of the lipase created for substrate assisted catalysis (paper II) and three different variants with mutations which decreased the volume of the active site (paper III) were evaluated. Enzyme kinetics for the different variants were measured and translated into activation energies for comparison of the approaches.

In papers IV and V three different enzymes were used for rapid analysis of enantiomeric excess and conversion of O-acylated cyanohydrins synthesized by a defined protocol. Horse liver alcohol dehydrogenase, Candida antarctica lipase B and pig liver esterase were sequentially added to a solution containing the O-acylated cyanohydrin. Each enzyme caused a drop in absorbance from oxidation of NADH to NAD+. The product yield and enantiomeric excess was calculated from the relative differences in absorbance.

In paper VI a method for C-terminal peptide sequencing was developed based on conventional Carboxypeptidase Y digestion combined with matrix assisted laser desorption/ionization mass spectrometry. An alternative nucleophile was used to obtain a stable peptide ladder and improve sequence coverage.

Place, publisher, year, edition, pages
Stockholm: KTH, 2010. viii, 62 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2010:12
Keyword
Candida antarctica lipase B, monoacylation of diols, kinetic resolution, thermodynamics in enzyme catalysis, enzyme engineering
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-12831 (URN)978-91-7415-663-8 (ISBN)
Public defence
2010-06-04, FD5, AlbaNova University Center, Roslagstullsbacken 21, Stockholm, 15:42 (English)
Opponent
Supervisors
Note
QC20100629Available from: 2010-05-24 Created: 2010-05-12 Last updated: 2010-06-29Bibliographically approved

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