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Sifting Proteins Out of the MuD: Advancing proteomics research for complex protein mixtures
Martha Walz
One of the main goals of proteomics, the large-scale study of proteins and their structures, is to identify proteins that interact with one another. To identify proteins amid complex mixtures, they first need to be separated. Gel-based methods are the most commonly used technologies for separating proteins, with the ability to separate hundreds to thousands of proteins with extremely high resolution. Once the proteins are separated, mass spectrometry (MS) is used to identify them.
Although gel-based methods are widely used, they have several drawbacks: difficulty detecting hydrophobic proteins that self-associate in the presence of water, difficulty detecting low-level proteins, long experiment duration, and the inability to be automated. In order to improve the separation and identification of proteins, especially in complex protein samples, techniques such as shotgun proteomics and multidimensional protein identification technology (MuDPIT) have sprung up.
Shotgun proteomics
MuDPIT begins with breaking the proteins in a mixture into their constituent peptides. The peptides are then separated using multiple LC steps. The peptides are then analyzed by MS and the results are fed into SEQUEST to identify the proteins. Image courtesy of John Yates, The Scripps Research InstituteClick to enlarge. |
Rather than trying to isolate single proteins in a mixture, shotgun proteomics is a collection of techniques that are bottom-up rather than top-down in approach. A sample of proteins is digested into their constituent peptides prior to separation. The peptides are then directly analyzed in a mass spectrometer, collecting tandem mass spectra. Each mass spectrum relates back to the protein from which the peptide came, so the collection of proteins that were present in the original mixture can be reconstructed. The MS/MS spectra are searched against a protein database using SEQUEST, which is distributed by Thermo Fisher Scientific. SEQUEST correlates uninterpreted tandem mass spectra of peptides with amino acid sequences from protein and nucleotide databases. It then determines the amino acid sequence and thus the protein(s) and organism(s) that correspond to the mass spectrum being analyzed. Using this approach, collections of proteins that are related can be identified in a single experiment, alleviating the need to have the proteins be separated and then sequenced or identified individually, thus creating a huge time and labor savings.
“The shotgun proteomic approach overcomes problems of working with intact proteins such as physical and chemical diversity,” says John Yates, Professor of Cell Biology and Head of the Proteomics Mass Spectroscopy Lab at The Scripps Research Institute, La Jolla, CA, and lead inventor of SEQUEST. “By reducing the mixture of proteins into a mixture of peptides, the physical and chemical diversity is reduced to a level where there are mature technologies for analysis (i.e., high performance liquid chromatography [HPLC] and MS). There are some limitations to the approach, particularly when looking at modifications, but for the most part it is very effective to identify proteins in reasonably complex mixtures.”
The shotgun-based methods are useful for conducting large-scale global (or discovery-based) analyses of protein mixtures. One particular technique that has gained popularity in recent years for identifying proteins in complex mixtures is MuDPIT.
Into the MuDPIT
MuDPIT is a shotgun proteomics technique for the separation and identification of complex protein and peptide mixtures. MuDPIT lets researchers identify every protein in a sample without prior knowledge of the sample’s protein content. It couples 2-D chromatography of peptides in mass spectrometry-compatible solutions directly to tandem mass spectrometry, allowing for the identifications of proteins from highly complex mixtures. MuDPIT can identify all proteins in a protein complex, but not in a whole cell. “It depends on the complexity of the mixture and the range of protein abundances,” explains Yates. “The solution is to use more levels of protein fractionation to create less-complex mixtures.”
MuDPIT begins with the digestion of proteins in a protein mixture into their constituent peptides. The peptides are then separated in multiple chromatography steps. As the peptides elute from the chromatography column, they are sprayed directly into a linear ion trap MS. The MS assigns each peptide a mass/charge ratio. The most intense peptide signals are then fragmented into a second MS-MS scan that assigns each peptide a “fingerprint.” The fingerprints are then fed into databases such as SEQUEST to identify the proteins.
MuDPIT has many advantages over gel-based methods. “We find it provides comprehensive answers with the least amount of labor and expense,” says Yates. In addition, the band broadening associated with many chromatographic steps is avoided, so the capillary can be placed directly into the ion source of a mass spectrometer, which maximizes sensitivity. MuDPIT is also able to generate large amounts of useful data in a shorter period of time than gel-based methods.
Although there are many advantages to using MuDPIT, it does not solve all of the problems associated with complex protein mixtures. “When you get to very complex mixtures of proteins with large differences in protein abundance, i.e., plasma, MuDPIT has not been very effective,” says Yates. “Trying to determine relative ratios of modifications on proteins is tricky using the shotgun approach or the MuDPIT approach.”
MuDPIT in action
MuDPIT is being used to identify proteins in whole proteomes and organelles in rats, mice, humans, and microscopic parasites, and it can be used on plants as well. “The most important issue is whether there is a genome sequence for the organisms, as that greatly facilitates data analysis,” says Yates.
There are many variations on the MuDPIT approach, such as running proteins on a gel and then cutting up the whole gel, in-gel digestion of the proteins, and liquid chromatography (LC)-MS-MS of the peptide mixtures. Another is off-line 2-D LC-MS-MS. “All of these are variations on the theme” of shotgun proteomics, says Yates.
“MuDPIT is particularly effective for the analysis of protein complexes and so it will be used for those types of analyses quite a bit. As people improve on the technology, it will be used for ever more challenging biological analyses.”
Martha Walz is the Managing
Editor of R&D Magazine. She may
be contacted at ChromatographyTechniques@advantagemedia.com.
Note: This article originally appeared in the January 2007 issue of R&D Magazine, a sister publication of Chromatography Techniques.
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