Metabolic Research

IROA® Biochemical Quantitation Kits

IROA® Technical Information Sheet


  • IROA® Technical Information Sheet
An innovative technology termed isotopic ratio outlier analysis (IROA) was developed to overcome the analytical challenges associated with current, stable isotope labeled compounds used in untargeted /targeted metabolic profiling. These challenges minimally pertain to analytical variance, artifactual peaks, and metabolite identification. The IROA method uses metabolic incorporation of heavy (95% 13C) and light (5% 13C) nutrients, such as D-glucose and other carbon energy sources, to give rise to unique labeling patterns that can be readily identified and distinguished by mass spectrometry. Such a method would be useful in interlaboratory quantitative evaluations, if widely available. 
 
Cambridge Isotope Laboratories, Inc. (CIL) is proud to offer a series of IROA® Biochemical Quantitation Kits for metabolic profiling of various cell populations and biological samples. 
 
These kits contain the protein reagents and tools necessary for the successful metabolic labeling, identification, and quantitation of metabolites in various cell populations. Refer to product list for details.
 

Kit Features and Benefits

  • Eliminates technical and analytical variance increases reliability
  • Removes artifacts and noise increases precision and determination of metabolome
  • Reproducible identification of knowns/unknowns
  • Accurate, relative quantitation
  • Automated solution (via ClusterFinder™ software)
  • Easy statistical interpretation of sample results
  • Broad applicability with experimental perturbations being user-defined

 

 

 

Resources

 

 

 

Frequently Asked Questions 

What does the IROA protocol involve?

In the basic IROA protocol, biomolecules in two cell populations – control and experimental – are randomly labeled with stable 13C (95% and 5% D-glucose 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.

What does IROA’s ClusterFinder™ software and portal provide?

The software automates peak identification and quantitation from the raw Mass Spectrometry data, while the portal provides assistance on data interpretation and statistical analysis. Further to the portal, it provides 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.

What are acceptable MS data formats for ClusterFinder™?

ClusterFinder™ directly supports the following Mass Spectrometry data file formats: mzXML & mzML, mzDATA. Note: all major instrument vendors provide software for converting their proprietary data files into one of the formats supported by ClusterFinder™. 

What are the requirements for running ClusterFinder™ software?

Hardware requirements:

1. Intel Pentium III /800 MHz or higher (or compatible); dual-core processor or higher;

2. 16 GB RAM minimum

Software requirements:

1. Windows XP (x64 and x84)

2. Windows 7 SP1 (x86 and x84)

3. Windows Server 2008 R2 SP1 (x64)

4. Windows Server 2008 SP2 (x86 and x84)

5. Windows 8

6. Windows Server 2012

Computer needs to be network-accessible.

How is the issue of sample-to-sample variance overcome with the IROA protocol?

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.

How many samples can be analyzed with IROA's 100 and 200 kits? Also, what is the per sample cost?

The supplied media in these biochemical quantitation kits is sufficient for a minimum of 48 experimental and 48 control cell sample analyses (maximum of 96 per sample type if only 0.5 mL is used per sample).  Procedurally, 0.5 mL of media/sample during the cell growth phase is sufficient for 5 cell doublings to assure full label incorporation.  As a general guideline, LC-MS injections of 7 or 12 µL for positive or negative ESI, respectively are recommended from a final volume of 400 µL in the 96-well plate.  This equates to approximately $ 15- 30 USD per experimental sample and includes the software for analysis .

 

References

Viant, M.R.; Kurland, I.J.; Jones, M.R.; Dunn. W.B. 2017. How close are we to complete annotation of metabolomes? Curr Opin Chem Biol, 36:64-69 PMID:28113135

Qiu, Y.; Moir, R.; Willis, I,; Beecher, C., et al. 2016. Isotopic Ratio Outlier Analysis of the S. cerevisiae Metabolome Using Accurate Mass Gas Chromatography/Time-of-Flight Mass Spectrometry: A New Method for Discovery. Anal Chem88, 2747-2754. PMID:26820234

Clendinen, C.S.; Stupp, G.S.; Ajredini, R.; Lee-McMullen, B., et al. 2015. An overview of methods using 13C for improved compound identification in metabolomics and natural products. Front Plant Sci, 6, 1-13. PMID:2637967

Edison, A.S.; Clendinen, C.S.; Ajredini, R.; Beecher, C., et al. 2015. Metabolomics and Natural-Products Strategies to Study Chemical Ecology in Nematodes. Integr Comp Biol, 55, 478-485. PMID: 26141866

Stupp, G.S.; Clendinen, C.S.; Ajredini, R.; Szewc, M.A., et al. 2013. Isotopic Ratio Outlier Analysis Global Metabolomics of Caenorhabditis elegans. Anal Chem17, 11858-11865. PMID: 24274725

de Jong, F.A.; Beecher, C. 2012. Addressing the current bottlenecks of metabolomics: Isotopic Ratio Outlier Analysis™, an isotopic-labeling technique for accurate biochemical profiling. Bioanalysis, 4, 2303-2314. PMID:23046270

Krista Backiel

Krista Backiel

Marketing Manager and Metabolomics Manager

Krista is responsible for managing and promoting products that are utilized in Metabolomics and Clinical/Diagnostic MS. She spends a lot of her time developing new products to assist customers in their diverse research efforts.

More
Andrew Percy, PhD

Andrew Percy, PhD

Senior Applications Chemist - Mass Spectrometry

Dr. Andrew Percy is the Senior Applications Chemist for Mass Spectrometry. His responsibilities minimally involve overseeing product development, identifying new product market opportunities, assisting in the analysis of products for MS ‘omics applications, and providing technical support to customers.

More

Related Products

Catalog No.     Description 

IROA-100-50 IROA 100 for Yeast/Fungi Metabolic Profiling Kit
IROA-200-50 IROA 200 for Bacterial Metabolic Profiling Kit
IROA-200-UL IROA 200 Unlabeled Bacterial Media
IROA-300-250 IROA 300 for Mammalian Metabolic Profiling Kit
IROA-300-UL IROA 300 Unlabeled Mammalian Media 
IROA-PHENO-95-300 IROA 300 for Phenotypic Metabolic Profiling Kit
IROA-FLUX-05-300 IROA 300 for Fluxomic Metabolic Profiling Kit 

13C biochemical quantitation kits for precise and accurate biochemical profiling.