Detection of crosslinks within and between proteins by LC-MALDI-TOFTOF and the software FINDX to reduce the MSMS-data to acquire for validation.

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...

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Autores principales:	Christopher A G Söderberg, Wietske Lambert, Sven Kjellström, Alena Wiegandt, Ragna Peterson Wulff, Cecilia Månsson, Gudrun Rutsdottir, Cecilia Emanuelsson
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/42df66698a444a6b81c119185324f8d0
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spelling	oai:doaj.org-article:42df66698a444a6b81c119185324f8d02021-11-18T07:15:12ZDetection of crosslinks within and between proteins by LC-MALDI-TOFTOF and the software FINDX to reduce the MSMS-data to acquire for validation.1932-620310.1371/journal.pone.0038927https://doaj.org/article/42df66698a444a6b81c119185324f8d02012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22723907/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Lysine-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.Christopher A G SöderbergWietske LambertSven KjellströmAlena WiegandtRagna Peterson WulffCecilia MånssonGudrun RutsdottirCecilia EmanuelssonPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 6, p e38927 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Christopher A G Söderberg Wietske Lambert Sven Kjellström Alena Wiegandt Ragna Peterson Wulff Cecilia Månsson Gudrun Rutsdottir Cecilia Emanuelsson Detection of crosslinks within and between proteins by LC-MALDI-TOFTOF and the software FINDX to reduce the MSMS-data to acquire for validation.
description	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.
format	article
author	Christopher A G Söderberg Wietske Lambert Sven Kjellström Alena Wiegandt Ragna Peterson Wulff Cecilia Månsson Gudrun Rutsdottir Cecilia Emanuelsson
author_facet	Christopher A G Söderberg Wietske Lambert Sven Kjellström Alena Wiegandt Ragna Peterson Wulff Cecilia Månsson Gudrun Rutsdottir Cecilia Emanuelsson
author_sort	Christopher A G Söderberg
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.
title_short	Detection of crosslinks within and between proteins by LC-MALDI-TOFTOF and the software FINDX to reduce the MSMS-data to acquire for validation.
title_full	Detection of crosslinks within and between proteins by LC-MALDI-TOFTOF and the software FINDX to reduce the MSMS-data to acquire for validation.
title_fullStr	Detection of crosslinks within and between proteins by LC-MALDI-TOFTOF and the software FINDX to reduce the MSMS-data to acquire for validation.
title_full_unstemmed	Detection of crosslinks within and between proteins by LC-MALDI-TOFTOF and the software FINDX to reduce the MSMS-data to acquire for validation.
title_sort	detection of crosslinks within and between proteins by lc-maldi-toftof and the software findx to reduce the msms-data to acquire for validation.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/42df66698a444a6b81c119185324f8d0
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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.

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