Development of quenching and washing protocols for quantitative intracellular metabolite analysis of uninfected and baculovirus-infected insect cells

• Published in: Methods (San Diego, Calif.) Calif.), 2012-03, Vol.56 (3), p.396-407
• Main Authors: Tran, Trinh T.B., Dietmair, Stefanie, Chan, Leslie C.L., Huynh, Hoai T., Nielsen, Lars K., Reid, Steven
• Format: Article
• Language: English
• Subjects: Adenine Nucleotides - metabolism; Adenosine Triphosphate - metabolism; Amino Acids - metabolism; Animals; Autographa californica; Baculovirus expression system; Cell Line; Chromatography, High Pressure Liquid; Helicoverpa armigera; Helicoverpa zea; Insect cell culture; Metabolomics; Nuclear polyhedrosis virus; Nucleopolyhedrovirus - genetics; Quenching; Spodoptera - cytology; Spodoptera frugiperda; Washing
• ISSN: 1046-2023; 1095-9130
• DOI: 10.1016/j.ymeth.2011.11.009

► Endometabolome analysis methodology developed for baculovirus-infected insect cells. ► Pluronic® F-68 added to cold saline protects cells during quenching and wash steps. ► One wash step is sufficient to remove medium-derived amino acids. ► One wash step following quenching gave good adenosine phosphate recoveries. Metabolomics refer to the global analysis of small molecule metabolites in a biological system, and can be a powerful tool to elucidate and optimize cellular processes, particularly when integrated into a systems biology framework. Determining the endometabolome in cultured animal cells is especially challenging, due to the conflicting demands for rapid quenching of metabolism and retention of membrane integrity, while cells are separated from the complex medium. The challenge is magnified in virus infected cells due to increased membrane fragility. This paper describes an effective methodology for quantitative intracellular metabolite analysis of the baculovirus–insect cell expression system, an important platform for the production of heterologous proteins and baculovirus-based biopesticides. These two applications were represented by Spodoptera frugiperda (Sf9) and Helicoverpa zea (HzAM1) cells infected with recombinant Autographa californica and wild-type Helicoverpa armigera nucleopolyhedroviruses (AcMNPV and HaSNPV), respectively. Specifically, an ice-cold quenching solution comprising 1.1% w/v NaCl and 0.2% w/v Pluronic® F-68 (NaCl+P) was found to be efficacious in preserving cell viability and minimizing cell leakage during quenching and centrifugation-based washing procedures (prior to extraction using cold 50% v/v acetonitrile). Good recoveries of intracellular adenosine triphosphate, total adenosine phosphates and amino acids were obtained after just one wash step, for both uninfected and infected insect cells. The ability to implement wash steps is critical, as insect cell media are metabolites-rich, while infected insect cells are much more fragile than their uninfected counterparts. Hence, a promising methodology has been developed to facilitate endometabolomic analysis of insect cell–baculovirus systems for bioprocess optimization.