Evaluation of cell lysis methods for platinum metallomic studies of human malignant cells.
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.
Tran
Mai Quynh Thanh
MQ
Department of Chemistry, Umeå University, S-901 87 Umeå, Sweden.
Nygren
Yvonne
Y
Lundin
Christina
C
Naredi
Peter
P
Björn
Erik
E
D016923:Cell Death / Q000187:drug effects; D002457:Cell Extracts / Q000737:chemistry; D002458:Cell Fractionation / Q000379:methods*; D045744:Cell Line, Tumor; D002469:Cell Separation; D002853:Chromatography, Liquid; D002945:Cisplatin / Q000494:pharmacology; D005615:Freezing; D006801:Humans; D055432:Metabolomics / Q000379:methods*; D009363:Neoplasm Proteins / Q000032:analysis; D009369:Neoplasms / Q000473:pathology*; D009995:Osmosis / Q000187:drug effects; D010984:Platinum / Q000494:pharmacology*; D015203:Reproducibility of Results; D013054:Spectrophotometry, Atomic
D002457:Cell Extracts; D009363:Neoplasm Proteins; D010984:Platinum; D002945:Cisplatin