The MFA Suite™ (Metabolic Flux Analysis Suite) is a comprehensive software toolkit designed to quantify the rates, or “fluxes”, of intracellular metabolic pathways. By systematically accounting for all extracellular carbon inputs and outputs and major intracellular pathways, the MFA Suite enables researchers to reconstruct detailed flux maps that reveal the dynamics of cellular metabolism. This powerful approach is essential for identifying pathway bottlenecks, elucidating network regulation, and tracking the flow or fate of carbon within biological systems, offering critical insights for applications in metabolic engineering, systems biology, and beyond.
The MFA Suite™ comprises three specialized applications: ETA (Elementary Transformation Analysis), INCA (Isotopomer Network Compartmental Analysis), and PIRAMID (Program for Integration and Rapid Analysis of Mass Isotopomer Distributions), each tailored to distinct aspects of metabolic flux analysis.
ETA: Elementary Transformation Analysis
ETA (Elementary Transformation Analysis) is a robust MATLAB-based software package for determining cell-specific metabolic rates and uncertainties from extracellular time-course data in exponentially growing cultures. Designed for quantitative metabolic phenotyping, ETA automates regression-based analysis of nutrient uptake, product formation, and growth dynamics using balance equations, supporting exponential or linear growth models while accounting for metabolite degradation (e.g., glutamine). It employs Gaussian error propagation for precise uncertainty estimation—superior for small sample sizes—and includes F-tests for model fit assessment, interactive data selection, and t-tests for inter-experiment comparisons. With an intuitive GUI for data import (from Excel or manual entry), visualization, and export, ETA delivers specific rates (e.g., glucose uptake, lactate excretion, amino acid fluxes) and yields in tabular/graphical formats, serving as essential inputs for advanced MFA or flux balance analysis to map intracellular metabolism under steady-state conditions.
INCA: Isotopomer Network Compartmental Analysis
INCA (Isotopomer Network Compartmental Analysis) is a pioneering MATLAB-based software package for isotopomer network modeling and metabolic flux analysis (MFA), uniquely capable of handling both steady-state and isotopically non-stationary (transient) labeling experiments. Leveraging the elementary metabolite unit (EMU) framework, INCA automates the generation and solution of mass and EMU balance equations for metabolic networks of arbitrary complexity, enabling accurate flux estimation from extracellular fluxes, pool sizes, and mass isotopomer measurements via mass spectrometry (MS) or nuclear magnetic resonance (NMR). Key features include simulation of isotope labeling dynamics, comprehensive statistical analyses for goodness-of-fit testing, parameter confidence intervals, and model identifiability; constraint-based network analysis (e.g., flux balance and coupling); and optimization of experimental designs through computational algorithms. With an intuitive graphical user interface (GUI), command-line access, and built-in parallelization, INCA is ideal for studying metabolic regulation in mammalian cells, photosynthetic organisms, and beyond, providing deeper insights into transient labeling scenarios where traditional tools fall short.
PIRAMID: Program for Integration and Rapid Analysis of Mass Isotopomer Distributions
PIRAMID (Program for Integration and Rapid Analysis of Mass Isotopomer Distributions) is a versatile MATLAB-based tool that streamlines the extraction, quantification, and statistical analysis of mass isotopomer distributions (MIDs) from mass spectrometry datasets in stable isotope labeling experiments. Vendor-agnostic and supporting formats like .cdf, .mzML, and .mzXML, PIRAMID automates peak detection via probabilistic methods, composite isotopic cluster integration with Savitzky-Golay smoothing, baseline correction, and noise estimation for high-accuracy MID calculations across low- or high-resolution MS/MS data. It handles single/dual labeling with isotopes (²H, ¹³C, ¹⁵N, ¹⁸O, ³⁴S), corrects for natural isotopic backgrounds using molecular formulas, and enables real-time visualization, normalization to average percent enrichments (APEs), group comparisons via t-tests, and export to .xls with descriptive statistics and plots. Featuring a user-friendly GUI and method file plugins for batch processing, PIRAMID is crucial for targeted metabolomics, providing reproducible MID inputs for flux analysis tools like INCA while accelerating high-throughput omics workflows.