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Research Feature: OSU's Mass Spectrometry Facility and Microbial Proteomics
The OSU Environmental Health Sciences Center and the Department of Chemistry operate a mass spectrometry facility with the capability to do proteomics work. Currently, most of their analyses focus on proteins from cultured cells, tissues and bodily fluids from mammals and humans. But, they have also done analyses on Vaccinia virus and some microbial organisms, for example, the marine microorganism SAR11, and they are happy to work with researchers interested in exploring the technique.
Mass Spectrometry Basics
Mass spectrometry is an analytical technique often used to identify unknown compounds. "The technique has many different applications and the instrumentation is quite diverse," says Claudia Maier, a chemistry professor affiliated with the OSU mass spectrometry facility, "But every mass spectrometer has three basic building blocks."
The last step is data analysis where information from the detector is converted into a mass spectrum, a diagram showing the intensity, or relative proportion, of ions with different mass-to-charge ratios. The spectrum is used to identify the compound.
"Bottom Up" Protein Mass Spectrometry
One technique that may be useful for subsurface biosphere studies is "bottom up" protein mass spectrometry. It is called "bottom up" because it identifies the peptides that make up proteins, rather than the whole proteins themselves. Sarah Sowell, a doctoral student in Molecular and Cellular Biology and member of Steve Giovannoni's lab, has used a "bottom up" approach to study protein function in SAR11, the ubiquitous marine microorganism. She is the first in their lab to do proteomics, so part of her emphasis has been on developing successful procedures for analyzing very small amounts of protein.
Sarah points out that one of the most critical parts of the analysis is the sample preparation. Her procedure involves filtering cells from her sea water media, lysing the cells to remove the proteins and then using different techniques to select the proteins of interest, for example from the cell membrane. She works with different protein separation techniques, such as gel electrophoresis and high-performance liquid chromatography, and often combines them in order to identify more proteins. Her last step is to digest the proteins into peptides using the enzyme trypsin.
Most of the proteomics analyses at OSU are done using tandem mass spectrometry or MS/MS. In this technique a set of mass spectrometers are linked together. The first instrument (MS1 in the figure above) separates the peptides and produces a spectrum of their mass-to-charge ratios and relative abundance. Then, specific peptides are selected and fragmented by collision with a gas such as Argon or Nitrogen. The next mass spectrometer (MS2 above) generates a spectrum of the mass-to-charge ratio and relative abundance of the fragments made from each peptide. The amino acid sequence of the peptide can be determined from this spectrum.
Currently at the OSU facility, a quadrupole time-of-flight mass spectrometer (QTOF) and a matrix-assisted laser desorption/ionization tandem mass spectrometer (MALDI TOF/TOF) are the most commonly used proteomics instruments. They use different ionization techniques and often provide complementary data when used to analyze the same sample. Sarah also uses an ion trap mass spectrometer at Pacific Northwest National Laboratory, in which all ions are trapped in a single chamber and MS1 and MS2 mass spectrometry is performed at discrete times instead of different locations. The OSU Mass Spectrometry facility will be adding a linear trap quadrupole - Fourier transform mass spectrometer (LTQ-FTMS) to their instrumentation infrastructure later this summer.
The data generated by the mass spectrometer is compared to information in a protein database. Software programs create theoretical spectra from the database by simulating the process of breaking apart the proteins into peptides and the peptides into ion fragments and calculating their theoretical mass. It then compares the spectra from the experimental data with the theoretical spectra and looks for matches. If matches are made for a number of peptides from a specific protein, then the protein is likely in the sample. Claudia Maier explains that the process might only identify a small percentage of the protein sequence, but that still may be sufficient to identify the protein with high confidence.
Both Sarah and Claudia point out that in addition to sample preparation, the database is the other critical component of the analysis. If the database doesn't contain sequence information for the protein of interest, the protein can't be identified. The OSU facility uses several publically available databases, which are continually being updated. It is also possible to create a custom database. Sarah uses a database that the Giovannoni team created from the SAR11 genome when she works with SAR11 cultures. For proteins that have not yet been deposited in databases, the OSU mass spectrometry facility can do de novo-sequencing, but Claudia cautions that this approach can be cumbersome and challenging.
The complexity of the data analysis process is another reason that protein separation at the beginning of mass spectrometry is so important. If the sample contains peptides from many different proteins, then it becomes harder to match them in the database and correctly identify the proteins. For this reason, it is easiest to work with a culture of one organism than with a mixed community. Sarah has done some analyses of mixed communities filtered from bulk ocean water samples and searched against a large database specific for marine microorganisms. She says, “When you are working with a mixed community, sample size has to be large because a greater diversity of proteins and peptides in an environmental sample means that the detection of any one of them becomes harder. Also, separation of the proteins before mass spectrometry becomes that much more important.”
Claudia Maier stresses the importance of study design and sample preparation and says, “If you are contemplating a preteomics study, you want to involve people who do mass spectrometry before you plan the study. Ideally, you would work with a team that includes someone who knows how to grow the culture, someone who is knowledgeable about protein isolation techniques, and someone who is familiar with mass spectrometry.”
Claudia recommends the OSU Mass Spectrometry facility home page as a place to gather background information. It contains contact information for all of the faculty and technical staff associated with the facility, information about instrumentation, services, and sample preparation and links to background reading material.
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