Metabolomics/Analytical Methods/Mass Spectrometry
General Description of Mass Spectrometry:
One of the main goals of metabolomics is to quantify changes in the concentrations of metabolites, and mass spectrometry offers a way to achieve this. Mass spectrometers can measure the masses and relative concentrations of molecules in a sample, making mass spectrometry an indispensable tool in metabolomics.
The technique involves ionizing the molecules to be analyzed and measuring the mass to charge ratio to determine the mass of each molecule present in the sample. There are four steps to the general process of mass spectrometry:
1.Ionization: Molecules are ionized to positive ions.
2.Acceleration: Ions are accelerated.
3.Deflection: Ions are introduced to a magnetic field and deflected in slightly different directions based on their unique masses and the amount of positive charge the ion has. Less mass and more positive charge both result in more deflection.
4.Detection: The ions are detected electrically.
Common types of mass spectrometry used in metabolomics include Matrix-assisted laser desorption/ionization MS (MALDI-MS), Tandem MS-MS, Ion Trap MS, liquid chromatography MS (LC-MS), and gas chromatography MS (GC-MS).
1. 13C-Labeled Gluconate Tracing as a Direct and Accurate Method for Determining the Pentose Phosphate Pathway Split Ratio in Penicillium chrysogenum
Main focus: The researchers used the fungus Penicillium chrysogenum in their attempt to accurately quantify the split ratio of 6-phosphogluconate between the oxidative and non-oxidative Pentose Phosphate Pathway (PPP). Previous studies attempting to quantify the split ratio in other organisms had results based on assumptions, but these people have overcome these difficulties with a relatively straight-forward approach. By feeding the cells simultaenously with trace amounts of 13C-labeled gluconate, which enters the pathway by directly being converted to 6-phosphogluconate, and using glucose free of any carbon label. They used a Quatro-LC triple quadrupole mass spectrometer to analyze the concentrations of the various intermediates in glycolysis and the PPP. They used a model formula to quantify the split ratio. They were able to assert that the PPP split ration was 51.1% with a 95% confidence interval, suggesting that the 13C-labeled gluconate tracing method is a much more sensitive and accurate method of determining flux which does not disturb the over all metabolism of the cell.
Terms: 1.Gluconate: a six carbon molecule with five hydroxyl groups terminating in a carboxylic acid group that can be converted to 6-phosophogluconate, then to fructose-6-phosphate, and used in glycolysis.
2.Isotopomer: Isomers have the same number of isotope atom but with different positions
3.Chemostat: A chemically static environment for growing microorganisms
4.Metabolic Flux Analysis (MFA): technique used for determining the rate at which a metabolite is produced in a metabolic process.
5.Liquid Chromatography Mass Spectrometry: Combines the physical separation capabilities of liquid chromatography with mass spectrometry. Generally used to find the composition of complex samples, such as those of biological origin.
Connection: The connection here with what we've learned in the course is obvious – they are trying to determine the flux of molecules between the oxidative and non-oxidative portions of the pentose phosphate pathway, which we learned about in chapter 14. This is the pathway that converts glucose-6-phosphate to ribose 5-phosophate, which can be used to synthesize nucleic acids and coenzymes. There is also a connection with what we learned about the regulation of metabolic pathways in chapter 15, where the rate of metabolic flow was introduced – this is flux, and is mentioned throughout the article.
2. Pathway Confirmation and Flux Analysis of Central Metabolic Pathways in Desulfovibrio vulgaris Hildenborough using Gas Chromatography-Mass Spectrometry and Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry
Main focus: The researchers used the sulfur-reducing bacterium Desulfovibrio vulgaris Hildenborough and tried to determine some information about its metabolism. GC-MS and FT-ICR MS were used to quantitate the amounts and types of metabolites the bacteria produced and to determine whether D. vulgaris's cytric acid cycle was oxidative or reductive (a fact that was previously unknown), and also to examine its glycolytic pathway and Pentose Phosphate Pathway. FT-ICR MS allowed them to confirm that the citric acid cycle in this sulfur-reducing bacterium was different from most aerobic organisms in that the citrate produced was the stereochemical opposite of the citrate found in most other organisms. This was confirmed with the help of 13C-labeling, similar to that used in the previous article. The experiments also enabled them to determine the flux rates along the pathways studied, which are annotated as part of figure 1 within the paper. All of this work was done to supplement the information already assumed about the organism from genomic analysis.
Terms: 1.Gas Chromatography-Mass Spectrometry (GC-MS) – Combines gas-exchange chromatography, in which the mobile phase is a gas, and mass spectrometry. Is incredibly specific and able to detect even trace amounts of a molecule in a sample.
2.Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) – Determines the mass to charge ratio of ions, which helps to determine the mass of particles.
3.Tricarboxylic Acid Cycle (TCA Cycle): Another name for the citric acid cycle.
4.Electrospray ionization (ESI) – Technique used in mass spectrometry to produce ions.
5.collision-induced dissociation (CID) – mechanism which fragments ions in the gas phase.
Connection: Their work ties in with the chapter about the citric acid cycle, chapter 16, because much of the work centered around determining how Desulfovibrio vulgaris's CAC worked based on what they knew of the genome and what enzymes they found not to be present in the organism, such as a lack of a citrate synthase homolog.
3. Inactivation of a Predicted Leader Peptidase Prevents Photoautotrophic Growth of Synechocystis sp. Strain PCC 6803
Main focus: The goal of this research was to determine the role of leader peptidase proteins LepB1 and LepB2 in the cyanobacterium Synechocystis sp. strain PCC 6803. Mutants of PCC were created by inserting a kanamycin cassette into the genome in the middle of LepB1 and LepB2. LepB2 mutants were impossible to procure, probably because the protein is essential for cell viability. LepB1 mutants' rates of photosynthesis decreased dramatically so that the cells were even harmed by high levels of light. The electron transport system continued to function, however, as long as cells were not exposed to high levels of light and only for short periods. The conclusion from this is that LepB1, which encodes a protein that removes signal peptides from precursor proteins that need to cross a membrane, actually plays a role in processing proteins that will eventually find their way in the thylakoid membrane. Mass spectrometry identified 8 proteins whose concentrations were always reduced in the mutants, four of which carried signal peptides, and also proteins not involved in the thylakoid system, suggesting that LepB1 can be found in both the thylakoid and plasma membranes.
Terms: 1.Peptidase: an enzyme that can break down a protein into single amino acids.
2.Signal/Leader peptides: short peptide chain that directs the post-translational transportation of a protein.
3.Photoautotroph – organism that synthesizes its “food” from inorganic substances using light as an energy source.
4.Phycobiliproteins – water-soluable fluorescent proteins in cyanobacteria and algae that capture light, the energy from which is used to fuel photosynthesis.
5.Western Analysis – Another name from the western blot, a method of detecting the presence of a specific protein in a sample.
Connection: Though we did not learn about the photosynthesis of cyanobacteria in the course, some general principles still apply. Decreasing the amount of active photosynthetic enzymes in the thylakoid membrane by decreasing the activity of the LepB1 gene product, which transports them there, will decrease the rate of photosynthesis
Metabolic Networks in Motion: 13C-based flux analysis http://www.nature.com/msb/journal/v2/n1/full/msb4100109.html
Summary: The main focus of this research is to study complex networks in metabolism through mathematical models that "attempt to monitor concentration changes of small chemical species within a cell" (par. 2). By using 13C-based flux analysis this study is able to "quantifies the integrated output of these component interactions" (par. 4).
Terms: Topology-the study of those properties of geometric forms that remain invariant under certain transformations, as bending or stretching. Moiety- an indefinite portion, part, or share. Stoichiometric- pertaining to or involving substances that are in the exact proportions required for a given reaction. Convergent- characterized by convergence; tending to come together; merging. Oxaloacetate - A four-carbon molecule found in the mitochondrion that condenses with acetyl CoA to form citrate in the first reaction of the Krebs cycle.
Relevance: This relates to what we have been studying in class because we have just begun to look at flux in metabolic system. Flux is the rate of turnover of molecules or enzymes through a metabolic pathway. And it is vital as it regulates the metabolic pathway's.
1. Scripps Center for Mass Spectrometry – METLIN metabolite database
Main focus: The aim of this site is to provide links to various resources of use to someone doing metabolomics research using mass spectrometry. There is a metabolite search function, an MS-MS search function, an LC-MS search function, and an FT- MS search function. There is also a downloadable program with the same functions as the site.
Terms: 1.MS-MS: Tandem Mass Spectrometry. Can perform multiple steps of mass selection or analysis
2.FT-MS: Fourier transform mass spectrometry – defined above in article #2.
3.Retention Range: period of time for which data should be available for inquiry.
(More new terms could not be found.)
2. Human Metabolome Database
Main focus: This site focuses on obtaining and providing information about various metabolites, including but not limited to Tandem MS data. From its name, the site obviously focuses on humans as the species of interest. It seeks to combine clinical and biochemical data.
Terms: 1.Biomarker – substance indicating a specific biological state.
2.Applet – software program that runs in the context of another program (ie, internet browser).
(More new terms could not be found.)
Main focus: The site focuses on providing an all-inclusive, high resolution resource of metabolic mass spectra. You can search through text, mass spec analysis method, peak, and source of the data. No specific organism is mentioned, so it may be assumed that the website includes metabolites from various organisms. The spectra data are mainly from Japanese research universities. They have over 13,000 spectra on file.
Terms: (None could be found.)
Connection (for all three databases):: Databases such as these are indespensible tools for biochemists. The results of a mass spectrometry experiment are useless unless one can decipher the meaning of each of the individual peaks on the graph. These databases provide mass spectra for hundreds or thousands of metabolites, allowing for identification and quantification of the molecules present in a sample. This data can then be used to determine the flux through pathways, changes in pathways, and more.
Wikipedia article on Mass Spec: http://en.wikipedia.org/wiki/Mass_spectrometry