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Method for the Affinity Purification of Covalently Linked Peptides Following Cyanogen Bromide Cleavage of Proteins
- Source :
- Analytical Chemistry. 81:9885-9895
- Publication Year :
- 2009
- Publisher :
- American Chemical Society (ACS), 2009.
-
Abstract
- The low resolution structure of a protein can sometimes be inferred from information about existing disulfide bridges or experimentally introduced chemical crosslinks. Frequently, this task involves enzymatic digestion of a protein followed by mass spectrometry-based identification of covalently linked peptides. To facilitate this task, we developed a method for the enrichment of covalently linked peptides following the chemical cleavage of a protein. The method capitalizes on the availability of homoserine lactone moieties at the C-termini of cyanogen bromide cleavage products which support selective conjugation of affinity tags. The availability of two C-termini within covalently linked peptides allows for the conjugation of two distinct affinity tags and thereby enables subsequent removal of unmodified peptides by tandem affinity chromatography. Here, we demonstrate the stepwise implementation of this method using a polyhistidine tag and a biotin tag for the selective two-step purification of covalently linked cyanogen bromide fragments from increasingly complex protein samples. The method is independent of the nature of the covalent bond, is adaptable to fully denaturing conditions, and requires only low picomole quantities of starting material.
- Subjects :
- chemistry.chemical_classification
Cyanogen
Homoserine
Peptide
Cleavage (embryo)
Combinatorial chemistry
Chromatography, Affinity
Analytical Chemistry
chemistry.chemical_compound
4-Butyrolactone
Biochemistry
Affinity chromatography
chemistry
Biotin
Azurin
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Cyanogen bromide
Cyanogen Bromide
Polyhistidine-tag
Peptides
Subjects
Details
- ISSN :
- 15206882 and 00032700
- Volume :
- 81
- Database :
- OpenAIRE
- Journal :
- Analytical Chemistry
- Accession number :
- edsair.doi.dedup.....b6e03fbd384932299abf9dda440378e4