13C-MFA

13C-MFA

Over the past two decades, metabolic engineering has played a crucial role in advancing chemical and pharmaceutical biotechnology. A quantitative understanding of intracellular metabolic reaction rates, that is, the fluxes can help scientists to comprehensively study cellular properties through metabolic engineering. In the fields of metabolic engineering, microbiology, biotechnology and cell culture research, it is important to calculate metabolic pathway fluxes accurately in biological systems. ¹³C metabolic flux analysis (¹³C-MFA) has become the major method for determining detailed metabolic flux distributions in living cells. Creative Proteomics has designed a protocol for ¹³C MFA that contains parallel labeling experiments, isotope labeling measurements, statistical analysis and optimal experimental design. This protocol is developed based on incubation of microorganisms grown in two (or more) parallel media with ¹³C-labeled glucose tracer or ¹³C-glutamine tracer, followed by gas chromatography-mass spectrometry (GC-MS) to detect isotopic labeling of amino acids, glycogen-bound glucose/glutamyl and RNA-bound ribose. Metabolic fluxes are then estimated, followed by a comprehensive statistical analysis to determine the fit and calculate confidence intervals for the fluxes.

Figure 1. Summary of current ¹³C-MFA studies on different organisms. (Guo, W.; et al. 2016)

Our Services

In general, mutated genes and signaling pathway dysfunction in cancer have been found to affect the regulation of glucose metabolism. In addition, glutamine has recently emerged as a key substrate to support cancer cell proliferation, and the quantification of its metabolic flux is essential to understand the mechanisms by which this amino acid is involved in the metabolic rewiring that sustains tumor cell survival and growth.

Creative Proteomics has developed a novel metabolic flux analysis platform to provide ¹³C-MFA service in a competitive fashion. We can offer a wide range of services to support all research and development activities.

¹³C-MFA Process

Our teams apply ¹³C-glucose and ¹³C-glutamine to label the central carbon metabolism of cell lines and analyze the ¹³C labeling during central carbon metabolism by tracking ¹³C-glucose or ¹³C-glutamine separately (parallel experiments)

• Experimental Design

We estimate intracellular fluxes from extracellular rate measurements (i.e., substrate uptake rate and product excretion rate) and isotope labeling measurements (e.g., MS, tandem MS, and NMR). Our team obtains accurate and precise estimates of in vivo metabolic fluxes from available data

• Metabolic Model Construction

During the construction of the metabolic model for C-MFA, we complete all enzymatic reactions and other processes that may affect the labeling of the measured metabolites

• Selection of Optimal Isotopic Labeling Measurements

Creative Proteomics supports gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). The advantages of our method are high sensitivity, sufficient to measure intracellular metabolites, good chromatographic separation

• Selection of Isotope Tracers

The correct choice of isotopic tracer is essential to improve the performance of C-MFA. Our experts are capable of choosing different isotope tracers for different parts of the metabolism to obtain the best throughput resolution

• Parallel Labeling Experiments (PLEs) for ¹³C-MFA

Creative Proteomics has developed PLEs as another complementary approach to improve the performance of C-MFA providing. In PLEs, two or more tracer experiments are performed in parallel by using different tracers in each experiment. The data are then integrated into the ¹³C-MFA by fitting all labeling measurements from multiple experiments to a single flux model

Figure 2. The scheme of ¹³C-Metabolic Flux Analysis (¹³C-MFA). (Guo, W.; et al. 2016)

Features of Our MFA Platform

• Developed based on the most updated knowledge of biology, bioinformatics and software development
• Widely applicable to a wide range of metabolic system
• Professional bioinformatics teams & personalized bioinformatics analysis services.
• Advanced instrument platform
• Integrated quantitative methodologies and comprehensive solutions for metabolomics

Based on high-performance quantitative techniques and advanced equipment, Creative Proteomics has constantly updating our metabolic flux analysis platform and is committed to offering professional, rapid and high-quality services of ¹³C-MFA at competitive prices for global customers. Our personalized and comprehensive services can satisfy any innovative scientific study demands, please contact our specialists to discuss your specific needs. We are looking forward to cooperating with you!

Reference

1. Guo, W.; et al. ¹³C-Metabolic Flux Analysis: An Accurate Approach to Demystify Microbial Metabolism for Biochemical Production. Bioengineering. 2016. 3(1): 3.