Dataset Information

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

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.

SUBMITTER: Soderberg CA

PROVIDER: S-EPMC3377668 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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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 A G CA Lambert Wietske W Kjellström Sven S Wiegandt Alena A Wulff Ragna Peterson RP Månsson Cecilia C Rutsdottir Gudrun G Emanuelsson Cecilia C

PloS one 20120618 6

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-acquisit ...[more]

PMID: 22723907

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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.

Project description:BackgroundThe measurement of catecholamines and their metabolites in either urine or plasma is an important diagnostic test used to exclude the presence of neuroendocrine tumours. Because of weak chromatographic retention and potential ion-suppression, reverse-phase LC-MSMS is not ideal for analysis of these polar molecules. Here, we investigate derivatisation by ethylation as an alternative approach.MethodsA simple and rapid method involving acetaldehyde and a reducing agent was used to convert urine free metanephrines and catecholamines, and their deuterated analogues as internal standards, to mono-ethyl or diethyl- derivatives. Using an Agilent 6460 triple-quadrupole mass spectrometer, precursor and product ion mass spectra were recorded to allow comparison of multiple reaction monitoring methods for both derivatised and non-derivatised analytes under reverse-phase LC-MSMS conditions with positive electrospray ionization.ResultsConversion of biogenic amines to less polar ethyl derivatives increased their mass and enhanced the intensity of their molecular ions and fragments. Ethylation also improved the chromatographic properties of the amines, with greater retention and elution from reverse-phase HPLC columns with a methanol or acetonitrile gradient. The signal response of tandem mass spectrometric detection was increased up to 50-fold for ethyl metanephrines compared to non-derivatised compounds. This increase allowed for the omission of solid-phase extraction of urine as a clean-up step prior to analysis. The 'dilute-derivatise-shoot' method maintained analytical performance with respect to between-run imprecision (CV < 6%) and accuracy in an external quality assurance program. Gender-related ranges for free metanephrines in early-morning spot urines, collected from adult patients, were similar using either derivatised or non-derivatised samples.ConclusionsThe LC-MSMS detection of free urine biogenic amines can be greatly enhanced by ethyl derivatisation, which is easy and rapid to perform. Advantages include improved chromatography and lower limits of quantitation, that negate the requirement for solid-phase clean-up of urine prior to analysis. A disadvantage is the potential toxicity of the derivatising agents used if they are not handled appropriately.

Dataset Information

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

Publications

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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