IROA^® Biochemical Quantitation Kits

In the basic protocol, biomolecules in two cell populations (control and experimental) are randomly labeled with stable ¹³C (95% and 5% labeled media for the control and experimental groups, respectively). After a defined incubation period, the experimental group is perturbed (through a chemical, genetic, or environmental stressor) before uniform mixing, sample preparation, and LC- or GC-MS analysis. For more information, please see the seminal reference by de Jong et al. in the list below and the experimental figure in the IROA^® Biochemical Quantitation Kits flyer.

The software automates peak identification and quantitation from the raw MS data, while providing tools for statistical analysis and interpretation. The integrated Assay Portal enables basic statistics (e.g., regressions and variances) and analysis (e.g., principal component, random forest, and correlation), along with summary plots (e.g., volcano, hybrid, and metabolic mapping) of the distributions.

ClusterFinder directly supports the following MS data file formats: mzXML, mzML, and mzDATA, although mzXML is preferable and the data should be centroided. Note: all major instrument vendors provide software for converting their proprietary data files into one of the formats supported by ClusterFinder. It must also be noted that although ClusterFinder does not currently support GC-MS data inputs, these kits have been applied to metabolomic GC-MS applications (see Qiu et al. reference in the list below).

Ion suppression, stemming from the variability of ionization efficiency, is one of the biggest problems facing mass spectrometry data interpretation. There is no sample-to-sample variance in the IROA datasets because the experimental and control samples are prepped and analyzed together. Further, since the protein standards and analytes are chemically identical and measured in an identical environment, they share identical ionization efficiencies making the measurements more accurate.

The labeling media supplied with these kits can minimally accommodate 46 experimental and 46 control cell sample analyses. Procedurally, using a six-well plate and 3 mL of media /sample/well/passage is sufficient for five cell doublings to assure full label incorporation. This assumes a total volume of 278 mL media (250 mL plus 28 mL dialyzed fetal bovine serum), 2.5 generations per passage or a total 6 mL per sample. As a general guideline, LC-MS injections of 2 or 4 μL for positive or negative ESI, respectively, are recommended from a final volume of 500 μL in the six-well plate. This equates to approximately $40 USD per experimental sample and includes the software for analysis. Note that for this minimalist discussion, there is sufficient volume in each well for both positive and negative analysis as well as HILIC or other analyses.