Method for the affinity purification of covalently linked peptides following cyanogen bromide cleavage of proteins.
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.
Shi
Tujin
T
Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, Canada.
Weerasekera
Rasanjala
R
Yan
Chen
C
Reginold
William
W
Ball
Haydn
H
Kislinger
Thomas
T
Schmitt-Ulms
Gerold
G
D015107:4-Butyrolactone / Q000031:analogs & derivatives / Q000737:chemistry; D001400:Azurin / Q000737:chemistry*; D002846:Chromatography, Affinity; D003488:Cyanogen Bromide / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D010455:Peptides; C088386:homoserine lactone; D001400:Azurin; D015107:4-Butyrolactone; D003488:Cyanogen Bromide