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Detection of Crosslinks within and between Proteins by LC-MALDI-TOFTOF and the Software FINDX to Reduce the MSMS-Data to Acquire for Validation.

Söderberg, Christopher LU ; Lambert, Wietske LU ; Kjellström, Sven LU ; Wiegandt, Alena; Peterson Wulff, Ragna LU ; Månsson, Cecilia LU ; Rutsdottir, Gudrun LU and Emanuelsson, Cecilia LU (2012) In PLoS One 7(6).
Abstract
Lysine-specific chemical crosslinking in combination with mass spectrometry is emerging as a tool for the structural characterization of protein complexes and protein-protein interactions. After tryptic digestion of crosslinked proteins there are thousands of peptides amenable to MSMS, of which only very few are crosslinked peptides of interest. Here we describe how the advantage offered by off-line LC-MALDI-TOF/TOF mass spectrometry is exploited in a two-step workflow to focus the MSMS-acquisition on crosslinks mainly. In a first step, MS-data are acquired and all the peak list files from the LC-separated fractions are merged by the FINDX software and screened for presence of crosslinks which are recognized as isotope-labeled doublet... (More)
Lysine-specific chemical crosslinking in combination with mass spectrometry is emerging as a tool for the structural characterization of protein complexes and protein-protein interactions. After tryptic digestion of crosslinked proteins there are thousands of peptides amenable to MSMS, of which only very few are crosslinked peptides of interest. Here we describe how the advantage offered by off-line LC-MALDI-TOF/TOF mass spectrometry is exploited in a two-step workflow to focus the MSMS-acquisition on crosslinks mainly. In a first step, MS-data are acquired and all the peak list files from the LC-separated fractions are merged by the FINDX software and screened for presence of crosslinks which are recognized as isotope-labeled doublet peaks. Information on the isotope doublet peak mass and intensity can be used as search constraints to reduce the number of false positives that match randomly to the observed peak masses. Based on the MS-data a precursor ion inclusion list is generated and used in a second step, where a restricted number of MSMS-spectra are acquired for crosslink validation. The decoupling of MS and MSMS and the peptide sorting with FINDX based on MS-data has the advantage that MSMS can be restricted to and focused on crosslinks of Type 2, which are of highest biological interest but often lowest in abundance. The LC-MALDI TOF/TOF workflow here described is applicable to protein multisubunit complexes and using (14)N/(15)N mixed isotope strategy for the detection of inter-protein crosslinks within protein oligomers. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
PLoS One
volume
7
issue
6
publisher
Public Library of Science
external identifiers
  • WOS:000305583300067
  • PMID:22723907
  • Scopus:84862499676
ISSN
1932-6203
DOI
10.1371/journal.pone.0038927
language
English
LU publication?
yes
id
c39954df-b531-4daa-a8b4-339956e6225c (old id 2859114)
date added to LUP
2012-07-06 14:42:26
date last changed
2016-10-13 04:26:36
@misc{c39954df-b531-4daa-a8b4-339956e6225c,
  abstract     = {Lysine-specific chemical crosslinking in combination with mass spectrometry is emerging as a tool for the structural characterization of protein complexes and protein-protein interactions. After tryptic digestion of crosslinked proteins there are thousands of peptides amenable to MSMS, of which only very few are crosslinked peptides of interest. Here we describe how the advantage offered by off-line LC-MALDI-TOF/TOF mass spectrometry is exploited in a two-step workflow to focus the MSMS-acquisition on crosslinks mainly. In a first step, MS-data are acquired and all the peak list files from the LC-separated fractions are merged by the FINDX software and screened for presence of crosslinks which are recognized as isotope-labeled doublet peaks. Information on the isotope doublet peak mass and intensity can be used as search constraints to reduce the number of false positives that match randomly to the observed peak masses. Based on the MS-data a precursor ion inclusion list is generated and used in a second step, where a restricted number of MSMS-spectra are acquired for crosslink validation. The decoupling of MS and MSMS and the peptide sorting with FINDX based on MS-data has the advantage that MSMS can be restricted to and focused on crosslinks of Type 2, which are of highest biological interest but often lowest in abundance. The LC-MALDI TOF/TOF workflow here described is applicable to protein multisubunit complexes and using (14)N/(15)N mixed isotope strategy for the detection of inter-protein crosslinks within protein oligomers.},
  author       = {Söderberg, Christopher and Lambert, Wietske and Kjellström, Sven and Wiegandt, Alena and Peterson Wulff, Ragna and Månsson, Cecilia and Rutsdottir, Gudrun and Emanuelsson, Cecilia},
  issn         = {1932-6203},
  language     = {eng},
  number       = {6},
  publisher    = {ARRAY(0xae4aa88)},
  series       = {PLoS One},
  title        = {Detection of Crosslinks within and between Proteins by LC-MALDI-TOFTOF and the Software FINDX to Reduce the MSMS-Data to Acquire for Validation.},
  url          = {http://dx.doi.org/10.1371/journal.pone.0038927},
  volume       = {7},
  year         = {2012},
}