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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.

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 < 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.

SUBMITTER: Lambert W

PROVIDER: S-EPMC3218361 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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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.

Lambert Wietske W Söderberg Christopher A G CA Rutsdottir Gudrun G Boelens Wilbert C WC Emanuelsson Cecilia C

Protein science : a publication of the Protein Society 20110818 10

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 p ...[more]

PMID: 21780214

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Project description:BACKGROUND:The presence of a wide range of bioactive organic pollutants in wastewater and municipal water sources is raising concerns about their potential effects on humans. Not surprisingly, various approaches are being explored that can efficiently degrade these persistent organic pollutants. Use of peroxidases has recently been recognized as a novel remediation approach that may have potential advantages over conventional degradation techniques. However, testing the abilities of different peroxidases to degrade diverse emerging pollutants is tedious and cumbersome. RESULTS:In the present study, we present a rapid and robust approach to easily test the degradability of 21 different emerging pollutants by five different peroxidases (soybean peroxidase, chloroperoxidase, lactoperoxidase, manganese peroxidase, and horseradish peroxidase) using an LC-MSMS approach. Furthermore, this approach was also used to examine the role of a redox mediator in these enzymatic degradation assays. Our results show that some of the organic pollutants can be easily degraded by all five of the peroxidases tested, whereas others are only degraded by a specific peroxidase (or when a redox mediator was present) and there are some that are completely resistant to degradation by any of the peroxidases tested (even in the presence of a redox mediator). The degradation of furosemide and trimethoprim by soybean peroxidase and chloroperoxidase, respectively, was investigated in detail by examining the transformation products generated during their degradation. Some of the products generated during enzymatic breakdown of these pollutants have been previously reported by others, however, we report many new transformation products. CONCLUSIONS:LC-MSMS approaches, like the one described here, can be used to rapidly evaluate the potential of different peroxidases (and redox requirements) to be used as bioremediation agents. Our preliminary result shows peroxidases hold tremendous potential for being used in a final wastewater treatment step.

Dataset Information

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

Publications

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

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