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Evaluation of cell lysis methods for platinum metallomic studies of human malignant cells
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Surgery.
Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Surgery.
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2010 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 396, no 1, 76-82 p.Article in journal (Refereed) Published
Abstract [en]

Three cell lysis methods-freeze-thaw, osmosis, and a chemical detergent-based method-were evaluated as sample treatment procedures for platinum metallomic studies of in vitro grown human malignant cells exposed to cisplatin. The lysis methods are relatively mild, resemble those commonly used in proteomic studies, and were selected because of the proven reactivity of platinum drug metabolites and indications that platinum in exposed cells and plasma is mainly associated with proteins. The chemical method gave an absolute lysis efficiency of greater than 80%, whereas the freeze-thaw and osmosis methods gave approximately 30% lower efficiency. The within- and between-batch lysis reproducibilities were, for all methods, better than 20 and 24% relative standard deviations, respectively. Total platinum concentration normalized to lysate protein content was statistically the same for all lysis methods. Reagents in the chemical lysis buffer did, however, react with platinum analyte compounds, making this method unsuitable for analysis of reactive compounds or for metallome profiling encompassing analytes with unknown reactivity. Of the lysis methods evaluated here, osmosis gave the highest cisplatin recovery, likely because this protocol is chemically inert and can be carried out at a constant low temperature. Therefore, it is the recommended cell lysis method for the determination of reactive and unknown intracellular platinum compounds.

Place, publisher, year, edition, pages
Elsevier , 2010. Vol. 396, no 1, 76-82 p.
Keyword [en]
Cell lysis, Sample preparation, Human malignant cells, Metallomics, Platinum, Cisplatin
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:umu:diva-29753DOI: 10.1016/j.ab.2009.08.044ISI: 000272406100011PubMedID: 19733145OAI: oai:DiVA.org:umu-29753DiVA: diva2:277927
Available from: 2009-11-23 Created: 2009-11-23 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Advances in analytical methodologies for studies of the platinum metallome in malignant cells exposed to cisplatin
Open this publication in new window or tab >>Advances in analytical methodologies for studies of the platinum metallome in malignant cells exposed to cisplatin
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Förbättrade analytiska metodologier för studier av platina-metallomet i maligna celler exponerade för cisplatin.
Abstract [en]

The scientific progress about the important chemotherapeutic drug substance cisplatin (CDDP) and its function has often been rendered by data difficult to interpret, and still many questions about its mode of action remains to be clarified by the scientific community. However, studies of CDDP possess a high complexity due to; i) low intracellular concentration, ii) many potential biomolecule targets, iii) poor or unknown stability of the intact drug and its biomolecule adducts and iv) complex and varying sample matrices. Metallomic studies, using advanced analytical techniques may contribute to clarify the interactions between CDDP and intracellular biomolecules. For a successful outcome sample preparation conditions as well as separation and detection techniques must be carefully selected and optimized to achieve accurate results and correct interpretation of data.

        This thesis describes some new and improved analytical methodologies for characterizing the Pt metallome in CDDP-exposed malignant cells. The developed methods are based on powerful liquid chromatography (LC) methods hyphenated to sensitive detection by inductively coupled plasma- (ICP) and electrospray ionization mass spectrometry (ESIMS). Consideration has also been taken about sample preparation conditions.

        By selecting “chemically inert” sample preparation (cell lysis by osmosis) and separation (using only nonreactive or no additatives) conditions we could avoid the formation of platinum artifact compounds previously described in the literature (Paper I and II). Using oxygen containing organic solvents with high boiling points (dimethylformamide; DMF, 1,4-dioxane, n-propanol and ethanol) as alternatives to acetonitrile in the LC separations, significant improvements were achieved in ICPMS sensitivity and robustness. When evaluated in combination with chromatographic performance and ESIMS detection the overall best performance was achieved with n-propanol (Paper II, III and IV). From the studies in Paper II we could show that free intact CDDP can be found in malignant cells, as supporting evidence for passive or endocytotic uptake of the drug and further estimate a half-life for intracellular CDDP to about 15 minutes. Such data has not been shown before. In Paper V, the above improved LC methods were used to demonstrate differences in the platinum and cupper metallome from sensitive and resistant T289 melanoma cells exposed to CDDP at near clinical levels.

        In a wider perspective we have shown the potential of using hydrophilic liquid interaction chromatography (HILIC) hyphenated to ICPMS detection as a general approach for analysis of hydrophilic metallo-compounds (Paper II). Taking advantage of the superior ICPMS performance using n-propanol gradients for reversed phase liquid chromatography (RPLC) possess a true alternative and /or complimentary technique to size exclusion chromatography (SEC) commonly applied within metallomic studies of biomolecules (Paper V). Using n-propanol in HILIC as well as in RPLC enables parallel detection by ICP- and ESIMS using only one set of chromatographic parameters (Paper III and IV), something commonly called for by scientists in the field.

Place, publisher, year, edition, pages
Umeå: Kemiska Institutionen, Umeå universitet, 2010. 8+44 p.
Keyword
Method development, Metallome, Inductively coupled plasma mass spectrometry, Electrospray ionization mass spectrometry, Liquid chromatography, Hyphenation, Organic modifier, Cisplatin, Platinum
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:umu:diva-37400 (URN)978-91-7459-085-2 (ISBN)
Public defence
2010-11-26, KBC-huset, Stora Hörsalen, KB3A1, Umeå Universitet, Umeå, 13:00 (English)
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Available from: 2010-11-05 Created: 2010-11-02 Last updated: 2010-11-05Bibliographically approved

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