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 the assessment of global oxidative stress. 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 the analysis of small molecule markers of cellular redox state.
Several indices of mitochondrial changes that occur in conditions of oxidative/nitrosative stress are carried out using state-of-the-art Seahorse technology on the XFe Flux Analyzer. Dr. Savita Sharma and Mr. Michael Alstott, MS have been trained in 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 certain cancer cells. Glycolysis can easily be determined on intact cells using the 96-well Seahorse instrument to determine cellular sequelae of oxidative/nitrosative stress. The Seahorse 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 XFe Analyzer.
Identification of proteins with differential expression is important to relate gene and protein concordance in cancer cells and to help identify key proteins that may play a role in cancer. Expression proteomics is conducted using state-of-the-art MS/MS orbitrap technology (Thermo Exploris 240 and Nano-LC instruments) that sequences the amino acids in tryptic digests of selected proteins. Expression proteomics also can be conducted in the RM-SRF using Reversed-phase Protein Array (RPPA), a state-of-art high throughput, quantitative proteomics technique that is compatible with SIRM (see below) - whereas all mass spectrometry-based proteomics is not compatible with SIRM. The RM-SRF has the world’s most comprehensive RPPA coverage for central metabolism which is the hallmark of metabolically reprogrammed cancers. Learn more about our Proteomics Core Facility.
Redox proteomics identification of oxidized proteins
Redox proteomics methods originated in the Butterfield laboratory. Principal redox proteomics approaches used include the identification of proteins with excess protein carbonyls, 3-nitrotyrosine, or protein-bound HNE. Identification of proteins with excess nitrosylation of cysteine residues also is 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 the identification of the amino acids on which the oxidative modification exists.
Proteomics to identify covalently modified proteins
Acetylation and methylation are two principal epigenetic-related 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” 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:
- Aerobic glycolysis: oxidation of glucose to pyruvate by NAD+
- Lactic fermentation: reduction of pyruvate to lactate
- Rate of glucose consumption normalized to measures of cell numbers or volumes
- Fraction of glucose consumed converted to excreted lactate
- Fractional enrichment of excreted lactate from 13C sources (e.g. glucose, Gln etc.)
- Quantification of free NAD+, NADH
- Pentose phosphate pathway: discrimination between oxidative (NADPH producing) and non-oxidative branches- ribose synthesis in ribonucleotide pool
- Serine/glycine pathways from 3PGA
- Gluconeogenesis from e.g. lactate or other glucogenic precursors
- Krebs cycle and anaplerosis (including via PC, and canonical and non-canonical glutaminolysis)
- Amino acid oxidation other than glutaminolysis (e.g. serinolysis, branched-chain amino acid oxidation into TCA, GDH, etc.)
- Nucleotide synthesis- purine and pyrimidine pathways
- Fatty acid oxidation
- Lipid synthesis and turnover (cf both acyl chain and glycerol headgroups from DHAP)- analysis of isotopologue distribution in most major lipids classes, >500 species
- Hexosamine pathway
- GSH synthesis and oxidation- GSSH/GSSG/GR cycle
- 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.
SIRM makes mass spectrometry-based proteomics INfeasible due to the massive isotopic labeling of proteins, however, RM-SRF provides RPPA proteomics services that ignore the isotopic labeling.