TY - JOUR
T1 - Method for the affinity purification of covalently linked peptides following cyanogen bromide cleavage of proteins
AU - Shi, Tujin
AU - Weerasekera, Rasanjala
AU - Yan, Chen
AU - Reginold, William
AU - Ball, Haydn
AU - Kislinger, Thomas
AU - Schmitt-Ulms, Gerold
N1 - Copyright:
Copyright 2010 Elsevier B.V., All rights reserved.
PY - 2009/12/15
Y1 - 2009/12/15
N2 - 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.
AB - 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.
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U2 - 10.1021/ac901373q
DO - 10.1021/ac901373q
M3 - Article
C2 - 19924875
AN - SCOPUS:72449172402
SN - 0003-2700
VL - 81
SP - 9885
EP - 9895
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 24
ER -