Skip to main content

Lund University Publications

LUND UNIVERSITY LIBRARIES

Thiol-exchange in DTSSP crosslinked peptides is proportional to cysteine content and precisely controlled in crosslink detection by two-step LC-MALDI MSMS

Lambert, Wietske LU ; Söderberg, Christopher LU ; Rutsdottir, Gudrun LU ; Boelens, Wilbert C. and Emanuelsson, Cecilia LU orcid (2011) In Protein Science 20(10). p.1682-1691
Abstract
The lysine-specific crosslinker 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP) is commonly used in the structural characterization of proteins by chemical crosslinking and mass spectrometry and we here describe an efficient two-step LC-MALDI-TOF/TOF procedure to detect crosslinked peptides. First MS data are acquired, and the properties of isotope-labeled DTSSP are used in data analysis to identify candidate crosslinks. MSMS data are then acquired for a restricted number of precursor ions per spot for final crosslink identification. We show that the thiol-catalyzed exchange between crosslinked peptides, which is due to the disulfide bond in DTSSP and known to possibly obscure data, can be precisely quantified using isotope-labeled... (More)
The lysine-specific crosslinker 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP) is commonly used in the structural characterization of proteins by chemical crosslinking and mass spectrometry and we here describe an efficient two-step LC-MALDI-TOF/TOF procedure to detect crosslinked peptides. First MS data are acquired, and the properties of isotope-labeled DTSSP are used in data analysis to identify candidate crosslinks. MSMS data are then acquired for a restricted number of precursor ions per spot for final crosslink identification. We show that the thiol-catalyzed exchange between crosslinked peptides, which is due to the disulfide bond in DTSSP and known to possibly obscure data, can be precisely quantified using isotope-labeled DTSSP. Crosslinked peptides are recognized as 8 Da doublet peaks and a new isotopic peak with twice the intensity appears in the middle of the doublet as a consequence of the thiol-exchange. False-positive crosslinks, formed exclusively by thiol-exchange, yield a 1:2:1 isotope pattern, whereas true crosslinks, formed by two lysine residues within crosslinkable distance in the native protein structure, yield a 1:0:1 isotope pattern. Peaks with a 1:X:1 isotope pattern, where 0 < X < 2, can be trusted as true crosslinks, with a defined proportion of the signal [2X/(2 + X)] being noise from the thiol-exchange. The thiol-exchange was correlated with the protein cysteine content and was minimized by shortening the trypsin incubation time, and for two molecular chaperone proteins with known structure all crosslinks fitted well to the structure data. The thiol-exchange can thus be controlled and isotope-labeled DTSSP safely used to detect true crosslinks between lysine residues in proteins. (Less)
Please use this url to cite or link to this publication:
author
; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
chemical crosslinking, DTSSP, disulfide, mass spectrometry, MALDI-TOF/TOF, protein structure, protein-protein interactions, small, heat shock protein, isotope pattern
in
Protein Science
volume
20
issue
10
pages
1682 - 1691
publisher
The Protein Society
external identifiers
  • wos:000295452000005
  • scopus:80053151544
  • pmid:21780214
ISSN
1469-896X
DOI
10.1002/pro.699
language
English
LU publication?
yes
id
2f3410c9-e5dc-4fd8-b5ec-2d722ab8d35b (old id 2175544)
alternative location
http://www.ncbi.nlm.nih.gov/pubmed/21780214?dopt=Abstract
date added to LUP
2016-04-01 10:07:25
date last changed
2022-01-25 19:57:18
@article{2f3410c9-e5dc-4fd8-b5ec-2d722ab8d35b,
  abstract     = {{The lysine-specific crosslinker 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP) is commonly used in the structural characterization of proteins by chemical crosslinking and mass spectrometry and we here describe an efficient two-step LC-MALDI-TOF/TOF procedure to detect crosslinked peptides. First MS data are acquired, and the properties of isotope-labeled DTSSP are used in data analysis to identify candidate crosslinks. MSMS data are then acquired for a restricted number of precursor ions per spot for final crosslink identification. We show that the thiol-catalyzed exchange between crosslinked peptides, which is due to the disulfide bond in DTSSP and known to possibly obscure data, can be precisely quantified using isotope-labeled DTSSP. Crosslinked peptides are recognized as 8 Da doublet peaks and a new isotopic peak with twice the intensity appears in the middle of the doublet as a consequence of the thiol-exchange. False-positive crosslinks, formed exclusively by thiol-exchange, yield a 1:2:1 isotope pattern, whereas true crosslinks, formed by two lysine residues within crosslinkable distance in the native protein structure, yield a 1:0:1 isotope pattern. Peaks with a 1:X:1 isotope pattern, where 0 &lt; X &lt; 2, can be trusted as true crosslinks, with a defined proportion of the signal [2X/(2 + X)] being noise from the thiol-exchange. The thiol-exchange was correlated with the protein cysteine content and was minimized by shortening the trypsin incubation time, and for two molecular chaperone proteins with known structure all crosslinks fitted well to the structure data. The thiol-exchange can thus be controlled and isotope-labeled DTSSP safely used to detect true crosslinks between lysine residues in proteins.}},
  author       = {{Lambert, Wietske and Söderberg, Christopher and Rutsdottir, Gudrun and Boelens, Wilbert C. and Emanuelsson, Cecilia}},
  issn         = {{1469-896X}},
  keywords     = {{chemical crosslinking; DTSSP; disulfide; mass spectrometry; MALDI-TOF/TOF; protein structure; protein-protein interactions; small; heat shock protein; isotope pattern}},
  language     = {{eng}},
  number       = {{10}},
  pages        = {{1682--1691}},
  publisher    = {{The Protein Society}},
  series       = {{Protein Science}},
  title        = {{Thiol-exchange in DTSSP crosslinked peptides is proportional to cysteine content and precisely controlled in crosslink detection by two-step LC-MALDI MSMS}},
  url          = {{http://dx.doi.org/10.1002/pro.699}},
  doi          = {{10.1002/pro.699}},
  volume       = {{20}},
  year         = {{2011}},
}