Method for the affinity purification of covalently linked peptides following cyanogen bromide cleavage of proteins

Tujin Shi, Rasanjala Weerasekera, Chen Yan, William Reginold, Haydn Ball, Thomas Kislinger, Gerold Schmitt-Ulms

Research output: Contribution to journalArticle

5 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)9885-9895
Number of pages11
JournalAnalytical Chemistry
Volume81
Issue number24
DOIs
StatePublished - Dec 15 2009

Fingerprint

Cyanogen Bromide
Purification
Peptides
Proteins
Availability
Affinity chromatography
Covalent bonds
Biotin
Disulfides
Mass spectrometry

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Method for the affinity purification of covalently linked peptides following cyanogen bromide cleavage of proteins. / Shi, Tujin; Weerasekera, Rasanjala; Yan, Chen; Reginold, William; Ball, Haydn; Kislinger, Thomas; Schmitt-Ulms, Gerold.

In: Analytical Chemistry, Vol. 81, No. 24, 15.12.2009, p. 9885-9895.

Research output: Contribution to journalArticle

Shi, T, Weerasekera, R, Yan, C, Reginold, W, Ball, H, Kislinger, T & Schmitt-Ulms, G 2009, 'Method for the affinity purification of covalently linked peptides following cyanogen bromide cleavage of proteins', Analytical Chemistry, vol. 81, no. 24, pp. 9885-9895. https://doi.org/10.1021/ac901373q
Shi, Tujin ; Weerasekera, Rasanjala ; Yan, Chen ; Reginold, William ; Ball, Haydn ; Kislinger, Thomas ; Schmitt-Ulms, Gerold. / Method for the affinity purification of covalently linked peptides following cyanogen bromide cleavage of proteins. In: Analytical Chemistry. 2009 ; Vol. 81, No. 24. pp. 9885-9895.
@article{baf0b564d9764d08868e5e8f1836a0a2,
title = "Method for the affinity purification of covalently linked peptides following cyanogen bromide cleavage of proteins",
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.",
author = "Tujin Shi and Rasanjala Weerasekera and Chen Yan and William Reginold and Haydn Ball and Thomas Kislinger and Gerold Schmitt-Ulms",
year = "2009",
month = "12",
day = "15",
doi = "10.1021/ac901373q",
language = "English (US)",
volume = "81",
pages = "9885--9895",
journal = "Analytical Chemistry",
issn = "0003-2700",
publisher = "American Chemical Society",
number = "24",

}

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

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.

UR - http://www.scopus.com/inward/record.url?scp=72449172402&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=72449172402&partnerID=8YFLogxK

U2 - 10.1021/ac901373q

DO - 10.1021/ac901373q

M3 - Article

C2 - 19924875

AN - SCOPUS:72449172402

VL - 81

SP - 9885

EP - 9895

JO - Analytical Chemistry

JF - Analytical Chemistry

SN - 0003-2700

IS - 24

ER -