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Thiol‐exchange in DTSSP crosslinked peptides is proportional to cysteine content and precisely controlled in crosslink detection by two‐step LC‐MALDI MSMS
Author(s) -
Lambert Wietske,
Söderberg Christopher A. G.,
Rutsdottir Gudrun,
Boelens Wilbert C.,
Emanuelsson Cecilia
Publication year - 2011
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.699
Subject(s) - thiol , chemistry , cysteine , isotope , yield (engineering) , stable isotope ratio , trypsin , lysine , peptide , amino acid , organic chemistry , biochemistry , enzyme , materials science , physics , quantum mechanics , metallurgy
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 [2 X /(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.