TECHNOLOGY FEATURE Pieces and parts 290 Blood work, with less sweat 290 Learning to love 2D 291 Box 1: Shaking the branches 290 Box 2: Looking for the hook-up 292 Taking on chromatography’s hard cases As protein chemistry becomes faster and more automated, a few tough problems in purification and analysis methods still remain. Incremental evolution in chromatography and revolutionary developments in other techniques are finally starting to lower some of the field’s highest hurdles. Alan Dove reports. Happy molecular biologists are all alike; every “many transmembrane proteins are highly .com/nature e protein chemist is unhappy in his own way. unstable after extraction and undergo con- Purifying nucleic acids, with their compara- formational changes and denaturation, even .natur tively uniform chemistry, simple building in the presence of detergents and stabilizing w blocks and predictable structures, is often agents.” tedious, but usually straightforward. In bio- Companies like Thermo Fisher are work- molecule purification, if you have seen one ing on new strategies, but for many scientists, http://ww nucleic acid, you have seen them all. Proteins, the quickest solution may be to drive around oup with their astonishing chemical and structur- the problem. “The chromatography tools r G al diversity, are a different story. If you have haven’t changed very radically. The radical seen one protein, you have seen one protein. ©iStockphoto.com/Gannet77 improvements are on the protein identifica- In the past few years, the rise of proteomics tion side,” says Speicher. In just a few years, Chromatography’s merry-go-round still in fashion. lishing from buzzword to bona fide discipline has he explains, mass spectrometers have evolved b simplified protein chemists’ lives consider- from distinguishing a few dozen proteins in Pu ably. Nonetheless, a few challenging classes of separation technologies recently1 (Box 2). a sample to distinguishing hundreds. The molecules still resist easy, large-scale purifica- New liquid chromatography columns from machines cannot quite handle a crude cell Nature tion. In a cruel twist, these hard cases include PhyNexus are built to fit standard pipettors, lysate yet, but biochemists can now obtain 7 many of the proteins biochemists and drug reducing sample volumes and shortening run clean data from surprisingly dirty samples. 200 developers are most interested in studying: times. High-performance liquid chromatog- Speicher recalls for example a project for © membrane- and organelle-bound signaling raphy (HPLC), long a workhorse of fast pro- which his group wanted to identify ligands molecules, post-translationally modified pro- tein purification, has been mechanized by GE that interacted with a specific membrane teins (Box 1) and scarce hormones diluted in Healthcare, with its automated AKTAxpress protein. Unfortunately, the researchers were complex biological fluids like serum. system. And Waters has upped the ante⎯or unable to get enough pure protein to analyze, Protein chemists have not given up, though, at least the pressure⎯even further, with just faint bands of potential ligands on a gel. and gradual improvements in chromatogra- ultra-performance liquid chromatography The team abandoned the project and took a phy and separations are chipping away at (UPLC) that promises to shorten processing different tack, “but today if we had that gel, some of the field’s longstanding problems. times and boost resolution. we’d just cut the band out and pop it in the Meanwhile, the rapid evolution of new analyti- “UPLC is something that's certainly on mass spec, and we’d have a protein identifica- cal technologies, especially mass spectrometry, the horizon,” says Gary Siuzdak, director tion, and if it turned out that it was a contam- is simplifying protein purification by allowing of the Center for Mass Spectrometry at the inant, we’d know it immediately, or if it was researchers to analyze dirtier samples. Scripps Research Institute. None of the new a specific ligand we’d get that identification technologies, however, are especially good at very early,” Speicher explains. The revolution will be ionized separating transmembrane proteins, a class Nonetheless, researchers need to be careful “If you talk about how [have] the chroma- that includes many of the most important not to overinterpret mass spectrometry data, tography and tools associated with chroma- signaling molecules in the cell. especially with impure samples. “You cannot tography improved, I think there have been The problem frustrates both biochemists compare it to what we have in nucleic acids. incremental improvements,” says David and product vendors. “Although we offer If you have a negative in a PCR, you can say Speicher, director of the proteomics facility a membrane-protein fractionation kit for ‘this mRNA is not expressed’. But if you can’t at the Wistar Institute. Although soluble- rapidly isolating these proteins from cells, we see something in the proteome, it may just protein purification has become relatively recognize that membrane proteins represent be below the limit of detection,” says Kai te straightforward, Speicher says membrane- a significant challenge for purification and Kaat, global business director for proteins protein purification has lagged behind. analysis,” says Greg Hermanson, director of at Qiagen. He adds that “from a proteomics Indeed, biochemists working on soluble technology at the Pierce division of Thermo technology standpoint, we still have a way proteins have been deluged with handy new Fisher Scientific. Hermanson adds that to go to really make a negative result not NATURE METHODS | VOL.4 NO.3 | MARCH 2007 | 289 TECHNOLOGY FEATURE But even with an expression system in hand, BOX 1 SHAKING THE BRANCHES purifying a membrane- or organelle-bound protein can be a nightmare. Biochemists For many proteomics projects, typically start by fractionating cells into their identifying the proteins in a complex cytosolic and membrane compartments, or compartment is only half the battle; using classical techniques like density-gradi- researchers also want to know exactly ent centrifugation. Unfortunately, the centri- how and when those proteins are post- fuge cannot distinguish one membrane from translationally modified. Tracking one another. of the most common and important As te Kaat explains, “If you look at mem- modifications, phosphorylation, has brane-bound proteins, what you usually get been a particularly tough challenge. in homebrew methods…is all membrane- methods Phosphoprotein antibodies or metal bound proteins, so this means you have affinity chromatography can isolate phosphorylated proteins, but neither endoplasmic reticulum, [and] you have the technique yields quantitative information organelles,” as well as plasma membranes. Worse, centrifugation techniques are notori- .com/nature about specific phosphorylation patterns. e Dendrimers shake the branches. (Courtesy of ously touchy, so results often vary from one Recently, though, researchers at the Ruedi Aebersold.) Institute for Systems Biology and the experiment to the next. .natur To address this problem, Qiagen now w University of Zurich solved this problem, using cleverly designed chemical reactions with off-the-shelf reagents2. To prove offers subcellular fractionation kits, includ- that the system works, the team tested it in human T cells. In one experiment, ing one specifically for plasma membranes. “What this kit actually delivers is a very, very http://ww they successfully identified all known tyrosine phosphorylation sites in the immunoreceptor tyrosine-based activation motifs of the T-cell receptor CD3 chains, high enrichment of the plasma membrane oup as well as previously unknown phosphorylation sites on 97 other proteins. with, for example, almost complete absence r G ⎯ of endoplasmic reticulum molecules,” says te The technique in which investigators use highly branched dendrimer structures to bind chemically modified phosphopeptides, then release and identify the peptides Kaat. The kits use proprietary buffers and col- by mass spectrometry⎯is not entirely new. "This goes back probably six or so umn resins, relying mostly on affinity purifi- lishing years that we started working on it," says Ruedi Aebersold, a professor at both the cation to isolate the desired components. b Institute for Systems Biology and the University of Zurich, and senior author on the Thermo Fisher also offers an extensive Pu paper. The original protocol was too hard for many researchers, though. "It was still series of kits and reagents for subcellular a very complicated procedure, and people who are not chemists had some difficulties fractionation. “We now allow researchers to Nature doing it," says Aebersold. fractionate cells into cytoplasmic, nuclear, 7 After several simplifications, the new method is considerably more user-friendly. "I membrane, mitochondrial, lysosomal or per- 200 would think that everyone who has good experimental skills on the lab bench would oxisomal fractions,” says Hermanson, adding © be able to do it," says Aebersold. He has discussed the possibility of commercializing that in many cases, the company's kits yield kits for the procedure, but with all of the reagents available from standard chemical intact organelles that can then be studied in vendors already, there is no need to wait. vitro or subjected to further purification. The New chemistry may soon come to the rescue for researchers working on proteins kits have been big sellers, and Thermo