news
RESEARCH PROFILES
Cells are miniature chemical factories. They use and generate energy, form proteins from amino acids, and break down complex carbohydrates. All of these reactions take place within a tiny volume, usually in the picoliter range. But “when we break open the cell, the kinetics are not favorable anymore because we’re moving from subpicoliter to often microliter volumes after lysis,” says Daniel Figeys. So when Figeys, Martin Ethier, and Weimin Hou at the Ottawa Institute of Systems Biology (Canada) and Henry Duewel (who is currently at Sigma-Aldrich Corp.) attempted to increase the efficiency of protein digestion for proteomics experiments, they looked to the cell for inspiration. “We said, ‘What if we try to recreate the environment that we observe in the cell in terms of concentration?’” he recalls. To better mimic the volume within a cell, Figeys and co-workers developed a proteomic reactor, which is described in this issue of JPR (pp 2754–2759). The reactor is an inexpensive microfluidic device in which proteins are preconcentrated, derivatized, and digested into peptides that can be analyzed by LC/MS/MS. In the current configuration, the reactor can bind up to 10–15 µg of protein. At the lower limit, the researchers detected proteins from a complex sample of cell lysate containing 0.5 µg of total protein and from a sample of 300 cells. Researchers can load complex protein mixtures or intact cells into the proteomic reactor, which contains a small amount of strong-cation exchange (SCX) material. Trypsin can be loaded separately or at the same time as the sample. To harvest proteins from cells, the researchers add lysis buffer. Initially, proteins bind to the SCX resin because of the low-pH environment within the reactor. After a few washes, reduction and alkylation reagents are added. When the researchers increase the pH of the system to 8, proteins are released from the SCX matrix, and trypsin becomes activated. The diges-
tion reaction proceeds in solution in a very small volume. Figeys says that the effective volume of the device is ~50 nL because the solution reaction is limited to the spaces between the SCX beads. After the proteins are digested, peptides are eluted with an LC/MS/MScompatible buffer.
DANIEL FIGEYS
Microfluidic proteomic reactor
Small wonder. In the tiny volume of the proteomic reactor, proteins are concentrated, reduced, alkylated, and digested.
But how does the proteomic reactor stack up against conventional techniques? To find out, the researchers put the device through its paces. As in conventional setups, most of the proteins that were reduced and alkylated in the reactor before digestion had better sequence coverage than those proteins that were only digested in the reactor. Therefore, solid-phase reactions with minute quantities of protein proceed as expected in the device. In a head-tohead comparison of the reactor with current gel-free methods, the reactor proved to be more sensitive. The researchers could detect 10× more unique proteins with the new device. The reactor was more sensitive “because we have a better concentration when we do the reaction, and we also can clean the sample better,” says Figeys. In addition, the number of steps is reduced when the proteomic reactor is used, so fewer
2500 Journal of Proteome Research • Vol. 5, No. 10, 2006
chances exist for sample loss, he says. According to Figeys, the whole process from cell introduction to peptide elution takes only 3–4 h, which is much faster than traditional methods that can take 4–12 h for digestion alone. The proteomic reactor concept really went against the grain at the time it was being developed, Figeys points out. For example, other scientists have generated microfabricated devices that incorporate multiple separation steps. Instead, Figeys and co-workers decided on a simpler system. “We wanted to create a device that would allow us to go directly from cells and couple that to MS. It’s a change in the way we think about processing samples,” he says. Because the microfluidic proteomic reactor is not microfabricated, it is easy to make. Also, the device is relatively inexpensive (~$1 each); in some settings, it is cheap enough to be used once and then thrown away. In addition, trypsin reactors in which the enzyme is immobilized were being reported when the group was refining the proteomic reactor. Figeys explains that he and his co-workers did not want to immobilize trypsin because of the risk that the enzyme might not face the right way or be able to move freely enough to digest proteins when bound to a support. Therefore, the researchers developed a system in which the digestion reaction occurred in solution. Figeys has big plans for the proteomic reactor. For example, single-cell analyses are in his sights. He says, “If we can go even smaller, we might be able to see what is happening overall from cell to cell when you look at their protein content.” Also, he envisions that the reactor will be useful for many applications beyond proteomics. Aside from the reduction, alkylation, and digestion reactions demonstrated in the current work, Figeys says that almost any reaction, such as fluorescent labeling, could be performed in the device. “I just hope other people will start using the reactor and that they’ll expand the types of reactions you can do,” he says. —Katie Cottingham
© 2006 American Chemical Society
