Technologies used by the RM SRF

Measures of oxidative/nitrosative stress
The RM SRF offers highly sensitive (ng of protein) immunochemical analyses of total oxidative and/or nitrosative stress. Dr. Butterfield, has published extensively on validating this method for assessment of global oxidative stress. If desired by MCC investigators, redox-coupled reduced glutathione and oxidized glutathione are determined by fluorescence or HPLC methods, and NAD(P)H and NAD(P)+ are determined by HPLC with electrochemical detection. In the rare event that equivocal results are obtained, MS-based detection of protein carbonyls will be employed to validate the immunochemical methods or LC-MS will be used for analysis of small molecule markers of cellular redox state. 

Mitochondrial biology
Several indices of mitochondrial changes that occur in conditions of oxidative/nitrosative stress are carried out using state-of-the-art Seahorse technology. Dr. Mitov trained in the Seahorse Bioscience company headquarters and has many years’ experience in designing, performing experiments and data analysis. Michael Alstott, MS, new to the RM SRF, also has been trained on use of the Seahorse instrument. The Warburg effect is a preponderance of energy production by glycolysis rather than through the mitochondrial electron transport chain in cancer cells. It can easily be determined on intact cells using the 96-well Seahorse instrument to determine cellular sequelae of oxidative/nitrosative stress. This platform allows dose-response studies to be carried out using mitochondrial biology as an endpoint. Determination of exo- and endogenous fatty acid utilization can be also investigated using the XF Analyzer.

Expression proteomics
Identification of proteins with differential expression is important to relate gene and protein concordance in cancer cells and to help identify key transcription factors that may play a role in cancer. Expertise in expression proteomics also exists in the RM SRF. Learn more at http://www.research.uky.edu/core/proteomics/location.html

Redox proteomics identification of oxidized proteins
A key RM SRF service, redox proteomics, originated in the Butterfield laboratory. Principal redox proteomics approaches used include identification of proteins with excess protein carbonyls, 3-nitrotyrosine, or protein-bound HNE. Identification of proteins with excess nitrosylation of cysteine residues is also available. Once identified, such oxidatively modified proteins are placed into their respective molecular pathways to determine cellular sequelae of oxidatively dysfunctional proteins and provide new research of importance in cancer. Moreover, MS/MS sequence analyses of tryptic peptides used in redox proteomics permit identification of the amino acids on which the oxidative modification exists.

Proteomics to identify covalently modified proteins
Acetylation and methylation are two principal covalent modifications to proteins with significant relevance to cancer. Proteomics identification of acetylated or methylated proteins is an important tool in the RM SRF.

Metabolomics
“Metabolomics” is the technical means to analyze metabolism by identifying and quantifying a large fraction of all of the metabolites present in a cell and how they change in response to perturbations of relevant metabolic networks. “Metabolomics” is not synonymous with “metabolism” because it is completely possible to carry out metabolomics analyses without gaining any insight, discovery, or understanding of metabolism.

Metabolomics requires appropriate metabolome coverage, and this in turn requires very high-end analytical instrumentation. Together, mass spectrometry and NMR are the most appropriate technologies worldwide. Informatics is the third critical component of metabolomics.

Metabolomics services fall into two broad categories, namely “profiling” and “stable isotope resolved metabolomics, or SIRM.” Profiling refers to targeted or untargeted experimental designs to determine the amounts of features in analytical platforms (e.g. different types of MS or NMR) or the identities and amounts of compounds in samples.

SIRM refers to the tracing of individual atoms from stable isotope-enriched source molecules through biochemical transformations into a variety of intermediates and products, for the purpose of pathway analysis and flux measurement 1-9.

In general, the choice of the metabolic source determines the biochemical network being probed. Listed below are a few of the thousands of possible probes:

  • [U-13C]-Glucose: survey 13C enters amino acids, nucleotides, lipids (glycolysis, PPP, CAC), hexosamine
  • 13C1, 13C2-Glc: discrimination between oxidative and non-oxidative branches of the Pentose Phosphate Pathway; PC anaplerosis
  • 13C15N Gln: glutaminolysis, nucleotide biosynthesis, energy metabolism (CAC), FA biosynthesis
  • 13C FA: b-oxidation, FA biosynthesis
  • 13C Ser: serine metabolism; 1-C metabolism; lipid metabolism
  • 13C glycerol: lipid backbone biosynthesis

The analytical platforms available include high resolution NMR, high-resolution (>400,000) high mass accuracy mass spectrometry (direct infusion or LC), GC-MS. These platforms provide information about the amounts and nature of metabolites and pathways and networks involved in central metabolism that can be probed using tracer experiments with combined isotopomer and isotopologue analysis, and multiple 13C/15N enriched precursors 1-9:  

1.   Aerobic glycolysis: oxidation of glucose to pyruvate by NAD+

2.   Lactic fermentation: reduction of pyruvate to lactate

3.   Rate of glucose consumption normalized to measures of cell numbers or volumes

4.   Fraction of glucose consumed converted to excreted lactate

5.   Fractional enrichment of excreted lactate from 13C sources (e.g. glucose, Gln etc.)

6.  Quantification of free NAD+, NADH

7.   Pentose phosphate pathway: discrimination between oxidative (NADPH producing) and non-oxidative branches- ribose synthesis in ribonucleotide pool.

8.   Glycogenolysis

9.   Serine/glycine pathways from 3PGA

10. Gluconeogenesis from e.g. lactate or other glucogenic precursors

11. Krebs cycle and anaplerosis (including via PC, and canonical and non canonical glutaminolysis)

12. Amino acid oxidation other than glutaminolysis (e.g. serinolysis, branched chain amino acid oxidation into TCA, GDH etc.)

13. Nucleotide synthesis- purine and pyrimidine pathways

14. Fatty acid oxidation

15. Lipid synthesis and turnover (cf both acyl chain and glycerol headgroups from DHAP)- analysis of isotopologue distribution in most major lipids classes, >500 species

16. Hexosamine pathway

17. GSH synthesis and oxidation- GSSH/GSSG/GR cycle

18. Lipid peroxidation

Generally speaking, there is no single marker per pathway in an embedded network, rather the combination of several isotopomers/isotopologues are needed for unambiguous assignments.

The specific isotopomer distributions also enable (in some circumstances) discrimination between the same biochemical reactions occurring in different compartments.  

The total number of named molecules including the lipids is >700. Including isotopologues and isotopomers it is several thousand. 

State-of-the-art mass spectrometry and NMR spectrometers tailored for applications in SIRM are used.

Useful Metabolomics Links



Page last updated: 4/5/2016 1:07:09 PM