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LC/MS/MS analysis of N-terminal protein adducts with improved sensitivity: A comparison of selected Edman isothiocyanate reagents.
Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
Stockholm University, Faculty of Science, Department of Applied Environmental Science (ITM).
2009 (English)In: International Journal of Analytical Chemistry, ISSN 1687-8779Article in journal (Refereed) Published
Abstract [en]

This study provides a basis for a new and straightforward method for LC/MS/MS-based screening of N-terminal protein adducts. This procedure is denoted the “FIRE procedure” as fluorescein isothiocyanate (FITC) gave superior sensitivity by LC/MS/MS when measuring adducts (R) of electrophilic compounds with a modified Edman procedure. The principles of the FIRE-procedure are that adducts to N-terminal amino acids selectively are detached and measured from of proteins after derivatisation by isothiocyanate Edman reagents. In this study, FITC, 4-N,N-dimethylaminoazobenzene 4′-isothiocyanate (DABITC) and 4-dimethylamino-1-naphthyl isothiocyanate (DNITC) were used to synthesize thiohydantoin analytes from valine and N-methylvaline. The sensitivity by LC/MS/MS was enhanced by up to three orders of magnitude as compared to phenyl isothiocyanate and higher as compared to pentafluorophenyl isothiocyanate. The FITC reagent will enable measurements of low background adduct levels. Synthesized analytes were characterised with, for example, 1H NMR, 13C NMR, LC/MS/MS, and UV.

Place, publisher, year, edition, pages
2009.
National Category
Chemical Sciences
Research subject
Environmental Chemistry
Identifiers
URN: urn:nbn:se:su:diva-35829DOI: 10.1155/2009/153472OAI: oai:DiVA.org:su-35829DiVA: diva2:288157
Note
Article ID 153472.Available from: 2010-01-20 Created: 2010-01-20 Last updated: 2011-10-10Bibliographically approved
In thesis
1. Methodology for hemoglobin adduct measurement: Fetal exposures to acrylamide and other genotoxic agents
Open this publication in new window or tab >>Methodology for hemoglobin adduct measurement: Fetal exposures to acrylamide and other genotoxic agents
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

There is increasing evidence that exposure to toxic chemicals during the prenatal period constitute a higher health risk than exposure during adulthood. To characterize exposure and identify risk factors, sensitive methods for analysis of chemicals in vivo with biomarker methods are needed. Adducts to hemoglobin (Hb) have been shown useful as biomarkers of dose in blood of reactive compounds/metabolites, which are toxic due to reactions with biomacromolecules.

The aim of this thesis was to develop a new method for the analysis of Hb adducts suitable for analysis of large sample series, and then to apply the method for measurements of Hb adducts from exposure to acrylamide, glycidamide and ethylene oxide in mother/cord blood samples from five European countries.

A new method for measurements of N-terminal Hb adducts, denoted the adduct FIRE procedure, was developed using the fluorescein isothiocyanate Edman reagent. With the new procedure, optimized for LC/MS analysis, a high sensitivity and reproducibility was achieved. The new method made it possible to perform measurements of low exposures to the studied genotoxic compounds in approximately 1350 maternal and cord blood samples.

The results show that the fetus is exposed to a similar in vivo dose of the studied compounds as the mother. The measured Hb adduct levels show that acrylamide exposure from food intake is higher for the participating mothers fromUK compared to the mothers from the other countries. The measured Hb adduct levels form a basis for evaluations of relationships between exposure and health risks, and ongoing studies indicate associations between acrylamide Hb adduct levels and birth weight.

The developed method was also used for identification of an unknown Hb adduct, which was shown to originate from methyl vinyl ketone (MVK), a highly reactive and toxic compound. The identity of the adduct was confirmed with synthesized standards. There exist both natural and anthropogenic sources to MVK, and to what extent the MVK-adduct reflects exogenous exposure is yet not clarified.

Place, publisher, year, edition, pages
Stockholm: Department of Materials and Environmental Chemistry (MMK), Stockholm University, 2011. 54 p.
National Category
Chemical Sciences
Research subject
Environmental Chemistry
Identifiers
urn:nbn:se:su:diva-63026 (URN)978-91-7447-376-6 (ISBN)
Public defence
2011-11-11, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (Swedish)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Epub ahead of print.Available from: 2011-10-20 Created: 2011-10-07 Last updated: 2011-10-10Bibliographically approved

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Rydberg, Pervon Stedingk, HansMagnér, Jörgen
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