title: Fast filtration sampling protocol for mammalian suspension cells tailored for phosphometabolome profiling by capillary ion chromatography - tandem mass spectrometry.
abstract: Capillary ion chromatography (capIC) is the premium separation technology for low molecular phosphometabolites and nucleotides in biological extracts. Removal of excessive amounts of salt during sample preparation stages is a prerequisite to enable high quality capIC separation in combination with reproducible and sensitive MS detection. Existing sampling protocols for mammalian cells used for GC-MS and LC-MS metabolic profiling can therefore not be directly applied to capIC separations. Here, the development of a fast filtration sampling protocol for mammalian suspension cells tailored for quantitative profiling of the phosphometabolome on capIC-MS/MS is presented. The whole procedure from sampling the culture to transfer of filter to quenching and extraction solution takes less than 10s. To prevent leakage it is critical that a low vacuum pressure is applied, and satisfactorily reproducibility was only obtained by usage of a vacuum pressure controlling device. A vacuum of 60mbar was optimal for filtration of multiple myeloma Jjn-3 cell cultures through 5μm polyvinylidene (PVDF) filters. A quick deionized water (DI-water) rinse step prior to extraction was tested, and significantly higher metabolite yields were obtained during capIC-MS/MS analyses in this extract compared to extracts prepared by saline and reduced saline (25%) washing steps only. In addition, chromatographic performance was dramatically improved. Thus, it was verified that a quick DI-water rinse is tolerated by the cells and can be included as the final stage during filtration. Over 30 metabolites were quantitated in JJN-3 cell extracts by using the optimized sampling protocol with subsequent capIC-MS/MS analysis, and up to 2 million cells can be used in a single filtration step for the chosen filter and vacuum pressure. The technical set-up is also highly advantageous for microbial metabolome filtration protocols after optimization of vacuum pressure and washing solutions, and the reduced salt content of the extract will also improve the quality of LC-MS analysis due to lower salt adduct ion formation.
authors.affiliation: Department of Biotechnology, NTNU Norwegian University of Science and Technology, Trondheim, Norway.
authors.forename: Hans F N
authors.initials: HF
authors.lastname: Kvitvang
authors.affiliation: Department of Biotechnology, NTNU Norwegian University of Science and Technology, Trondheim, Norway. Electronic address: Per.Bruheim@ntnu.no.
authors.forename: Per
authors.initials: P
authors.lastname: Bruheim
mesh_terms: D045744:Cell Line, Tumor; D002477:Cells / Q000737:chemistry* / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D005374:Filtration / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D055432:Metabolomics / Q000379:methods*; D010750:Phosphoproteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D010750:Phosphoproteins
keywords: Capillary ion chromatography; Fast filtration; Mammalian suspension cells; Sampling protocol; Tandem mass spectrometry