Search bioRxiv⌕ Search

Biology subjects

Timpen, L. E.

Publications and source records attributed to Timpen, L. E..

2 recordsLinked to original sources

Collective parameter estimation of related models with an initial stability constraint

Parameterisation of dynamic biochemical network models is a challenging aspect of systems biology. Especially when the parameter space is large and data is semi quantitive but comparable across different experimental conditions. Here, we present a set of command line tools utilising Pycotools (COPASI) that leverages the power of high-performance computing to facilitate parameter estimation of large models with many unknown parameters. In particular, we expand upon the abilities of Pycotools to address two particular issues. Firstly, the difficulty of constraining a models parameterisation to assume the system begins in a steady state (prior to a perturbative stimulation). And secondly, parameterising against relative quantitative time series data that have no absolute scale. Our software operates on the SLURM workload manager system and can be applied to any parameter estimation against time series data produced by applying a single perturbation at time zero to an equilibrated system. We validate that our technique can produce a parameterised model of the MTOR (mechanistic target of rapamycin) network based on semi-quantitative time-series data from 2 breast cancer cell lines, stimulated with insulin and amino acids. We also show our model can make reasonable predictions on distinct signaling dynamics in one breast cancer cell line based on the other by adjusting the initial protein quantities only. In conclusion, models should fit both the initial steady state and the dynamics following stimulation, given that stabilising systems prior to stimulation is a common experimental protocol in signaling research. By expanding standard tools, commonly used in the field, we have developed a widely applicable method, which can easily be evaluated and is amenable to wide general use.

systems biology↗

Automated liquid handling extraction and rapid quantification of underivatized amino acids and tryptophan metabolites from human serum and plasma using dual-column U(H)PLC-MRM-MS and its application to prostate cancer study.

Free amino acids (AAs) and their metabolites are important building blocks, energy sources and signaling molecules associated with various pathological phenotypes. The quantification of AA and tryptophan (TRP) metabolites in human serum and plasma is therefore of great diagnostic interest. Robust and reproducible sample extraction and processing workflows as well as rapid, sensitive absolute quantification of AA and TRP metabolites are required to identify candidate biomarkers and to improve current screening methods. We developed a validated semi-automated extraction and sample processing workflow using a robotic liquid handling platform and a rapid method for the absolute quantification of 20 free, underivatized AAs and 6 TRP metabolites using dual-column U(H)PLC-MRM-MS. The automated extraction and sample preparation workflow is designed for use in a 96-well plate format, allowing robust and reproducible high sample throughput without the need for further SPE, evaporation and/or buffer exchange. Samples extracted from serum and/or plasma in 96-well plates can be transferred directly to the U(H)PLC autosampler. The dual-column U(H)PLC-MRM-MS method, using a mixed- mode reversed-phase anion exchange column with formic acid as mobile phase modifier and a high- strength silica reversed-phase column with difluoroacetic acid as mobile phase additive, provided absolute quantification with nanomolar lower limits of quantification (LLOQ) for all metabolites except glycine (LLOQ: 2.46 {micro}M) in only 7.9 minutes. The semi-automated extraction workflow and dual-column U(H)PLC-MRM-MS method was applied to a human prostate cancer study and was shown to discriminate between treatment regimens and to identify amino acids responsible for the statistical separation between healthy controls and prostate cancer patients on active surveillance.

biochemistry↗