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.1 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 software)
  • Easy statistical interpretation of sample results
  • Broad applicability with experimental perturbations being user-defined

 

 

 

 

 

 

 

Frequently Asked Questions 

What does the IROA protocol involve?

In the 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 (see workflow schematic on right).

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

The software automates peak identification and quantitation from the raw Mass Spectrometry data (see screenshot on right), 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, netCDF, Agilent CSV export, and Thermo Raw. 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 normalization achieved in the IROA datasets?

Spectral TIC normalization is achieved using the MS total useful signal (MSTUS) approach (developed by Warrack, B.M. et al. 2009. Chromatogr B Analyt Technol Biomed Life Sci, 877, 547–552). In this approach, only the components that are common to all signals are used after baseline correction and removal of the artifacts (from xenobiotics or chemical noise, for instance) and nonbiological compounds that carry no IROA signatures.

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.

According to the protocol, amino acid mixes are to be added to the bacterial and mammalian kits. How does one distinguish the amino acids that are added to the media as standards from the ones that are metabolized from glucose?

The purpose of the media is to provide the necessary nutrients for growth and reproduction of multiple cell types. This will allow complete metabolic labeling (i.e., the 12C content is converted to either 5% or 95% 13C from D-glucose) after five cell doublings. The amino acids that supplement the media are 99% 13C. If a researcher is interested in following particular precursor(s) through biochemical pathways to distinguish one or more SILAC amino acids from amino acids metabolized from glucose, then the IROA Fluxomic protocol should be employed. In this protocol, cells are fully labeled with 5% 13C IROA media, while 99% 13C-labeled precursors are fed to the cells. During a time course, samples are taken, with the fate of the precursor(s) determined with the ClusterFinderTM software.

How many samples can be analyzed with IROA's 100 and 200 (for yeast/fungi and bacterial metabolic profiling)​ biochemical quantitation kits? Also, what is the per sample cost?

The supplied media in these 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:

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 [PubMed]

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:26379677 [PubMed]

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 [PubMed - in process]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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.

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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.

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

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