Courtesy of John Yates III
In the postgenomic era, there are the haves and the have-nots. Some resources, such as the genome sequence data that underlies functional genomics, are available to all. But mass spectrometry (MS), the signature tool for proteomics and a focus of my own research, remains mostly in the hands of the haves.
Traditionally large, complex, and expensive, mass spectrometers are often found not in individual labs but instead in university core facilities. Prices are dropping to the point where a small group of scientists could pool resources to acquire one. Yet researchers continue to send their samples to the core for analysis, or collaborate with proteomics specialists willing to process the samples for them.
Such unions are certainly fruitful, bringing proteomics prowess to labs that otherwise lack the skill, and boosting the publication records of the mass spec specialists. But this paradigm is insufficient in the...
TOWARD A SUBCELLULAR COMPENDIUM
MS is so valuable to functional genomics because it is unbiased. Microarray and RNA interference experiments must be targeted to specific genes or sequences. But MS is different: It reports data regardless of whether the experimenter is aware of a particular protein's existence. The resulting mass data can be used as a key to look up protein addresses in sequence databases and thereby identify them.1
The proteins don't even need to be purified; using tandem MS and a process called shotgun proteomics (see figure), it is possible to directly identify the protein components present in complex mixtures.23 These features, combined with frequent increases in the speed, sensitivity, and throughput of MS hardware, make the technique particularly amenable to the large-scale analysis of proteins in cells and subcellular compartments that is the essence of proteomics.
For several years my lab has been working in collaboration with researchers, both at Scripps and elsewhere, to apply the power of MS to the characterization of cellular structures and organelles. Subcellular fractionation is a messy and difficult business to be sure, but progress has been made. The two studies described below emphasize the potential value of mass spectrometers in the hands of biologists.
Courtesy of John Yates III
The first study involves the nuclear envelope (NE), the bimem-branous fence that separates the cytoplasm from the nucleus. Thirteen human diseases have been associated with disruptions of proteins integral to or interacting with the NE and prior analyses have identified 13 integral membrane proteins.4 Eric Schirmer and colleagues at the Scripps Research Institute used a subtractive proteomics method to conduct a more thorough analysis.5
The project was particularly challenging because the outer membrane is contiguous with, and shares properties with, the endoplasmic reticulum (ER), making it difficult to know which proteins are specific to the nuclear envelope and which are specific to the ER. Schirmer's team isolated microsomal membranes containing ER and mitochondria from rodent tissues, and performed an exhaustive proteomic analysis of the recovered proteins. NEs were then isolated, extracted with two different strategies to ensure the sample was enriched in integral membrane proteins, and finally subjected to MS.
Subtraction of the datasets revealed 67 new, integral membrane proteins. The human homologs of these proteins were identified and a representative set of the cDNAs representing the protein sequences were then epitope-tagged and localized within HeLa and COS7 cells. All the proteins tested localized to the NE, and at least five were found to strongly interact with the lamina, a polymer of intermediate-filament-type lamin proteins that line the inner nuclear membrane. As defects in lamin have been associated with different dystrophies and laminopathies, interactions between proteins and the lamina are of considerable interest.
Twenty-three of the proteins' genes colocalized to specific regions of the human genome associated with diseases that currently lack a known causative gene. Such associations are traditionally made through human genetic analysis, a long and tedious process. Admittedly, as each chromosomal region can be quite large and may contain several hundred genes, this analysis doesn't directly implicate any of these proteins in the disease. But it does make them interesting suspects.
Schirmer's experiment was made possible by combining clever cell biological techniques with proteomic ones. On the cell biology side, the team used known fractionation techniques to remove likely protein contaminants. The analysis of integral membrane proteins, meanwhile, was made possible by shotgun proteomics, a method in which proteins are proteolytically digested prior to MS analysis rather then being isolated biochemically.
The second illustrative study adopts a different tack. Instead of isolating an organelle, this study identified proteins involved in specific cellular events. Ahna Skop led a team of scientists, from the University of California at Berkeley and Scripps, that was interested in identifying proteins involved in cytokinesis, the process used to separate a mother cell into two daughter cells during mitosis.6 In late stages of cytokinesis a structure called the midbody forms, which contains microtubules tightly bundled by the cleavage furrow. Skop's team isolated this structure from Chinese hamster ovary cells and analyzed its components proteomically.
The group performed four separate enrichments, pooling the results from each analysis. Proteins were segregated by bioinformatic analysis to identify likely proteins involved in cytokinesis and their roundworm (
The majority of knockdowns (88%) resulted in observable defects. Seventeen proteins colocalized with tubulin, and all that were tested colocalized with the midbody. Because cytokinesis is highly conserved, these results will help researchers understand the process in other species.
THE DEMOCRATIZATION OF MS
Though strategically distinct, both the Schirmer and Skop studies illustrate a common denominator of state-of-the-art proteomics studies: different groups with different expertise collaborating on a specific project. Is this the right approach for biological studies – to have experts in proteomics technology working with biologists? Or can proteomics technology, in particular MS, be placed directly into biologists' hands?
Though mass spectrometers are complex instruments, operation by nonexperts is possible in a highly motivated laboratory. Dissemination of proteomics technology to nonexperts is complicated, however, by the fact that the technology evolves rapidly, with new developments appearing almost monthly. Proteomics practitioners are often both technology developers and adopters in pursuit of solutions to particular biological problems – for example, identifying the many posttranslational modifications that can occur on proteins simultaneously, as well as how those modifications change over time.
At the moment these experiments are technically challenging and thus difficult for nonexperts to execute, even when they have access to the necessary technology. Cutting-edge biology moves faster when experts collaborate, necessitating the need for proteomic centers of excellence, which ease the use of state-of-the-art equipment and methods far better than do, say, university core facilities.
For proteomics to flourish and significantly influence biology, several conditions must be met. Technology development must continue and be well funded. Experts in proteomics must collaborate with biologists to help solve real-world problems, thereby stretching the technology in new directions. New technologies and methods must be refined, made robust and reproducible, disseminated, and where appropriate, commercialized.
Eventually the technology will trickle down as it becomes more refined, and thus will become easier for nonexperts to use. And as more and more "lay" biologists see that they, too, can do MS research, others will surely follow.
The development of innovative and robust technologies for proteomics must democratize large-scale analyses rather than preserve them for the elite. MS technology should be placed into the hands of biologists. There is just too much new biology to be discovered for this not to happen.
John Yates III is a professor of cell biology and head of the Proteomic Mass Spectrometry Lab at the Scripps Research Institute in La Jolla, Calif. Trained at the University of Virginia and California Institute of Technology, Yates was the lead inventor of the SEQUEST software and the shotgun proteomics method. He has authored or coauthored more than 250 scientific publications.