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MEETING NEWS 17th International Symposium on Microscale Separations and Capillary Electrophoresis—Salzburg, Austria
Katie Cottingham reports from the
Protein digestion on-line with CE/MS
easily and that pepsin would cleave the proteins as they passed by. Although a coated monolith was a good idea, it was a difficult one to execute. “The biggest challenge was to fabricate the pepsin-containing thin film on the monolith [while] retaining the pepsin activity for a long period,” Kato says. The group attempted many different thin-film compositions, as reported in Analytical Chemistry (2004, 76, 1896– 1902). In the end, they found that 25% (w/v) pepsin in a 150-mM phosphate buffer gave the best results. Kato and co-workers have tested several proteins on the new column, which can be used 30 times over a period of 1 week. But the group is not resting on its laurels. “Now we are trying to coat monoliths with other enzymes,” says Kato, who adds that they are also further optimizing the on-line protein digestion CE/MS system. COURTESY OF MASARU KATO
To incorporate protein digestion on-line with CE and MS, Masaru Kato and coworkers at the University of Shizuoka, the Japan Science and Technology Corp., Ajinomoto Co. (all in Japan), and Stanford University developed a column containing a pepsin-coated monolith.
Scanning electron micrograph of a monolithic column coated with a pepsin sol–gel.
In past experiments, the researchers prepared an on-line enzyme reactor with trypsin encapsulated in a hydrogel. Small peptides were digested, but proteins were not. “It was assumed that proteins could not get through the hydrogel matrix,” says Kato. The group started exploring other ways to digest proteins on-line. One idea was to coat a macroporous photopolymerized sol–gel monolith with a thin film containing pepsin. They switched to pepsin because trypsin does not work in the acidic buffer that provided the best protein separation during CE. The researchers predicted that proteins would be able to move through this column 116 A
A new alternative to ICAT Pier Giorgio Righetti and colleagues at the University of Verona (Italy) say that the oft-cited quantitative proteomics strategy of isotope-coded affinity tag (ICAT), which labels –SH groups on cysteines, is no panacea. “ICAT cannot be used in proteomics studies with 2-D maps,” says Righetti. Originally optimized for LC/MS/MS methods, ICAT is incompatible with thiourea, which is a common solubilizing agent for 2-D gel electrophoresis. In fact, his group has demonstrated in earlier experiments that thiourea is a scavenger that destroys iodinated compounds, such as the ICAT reagent (Electrophoresis 2001, 22, 2066–2074). Another problem is that the alkylation reaction, whereby the ICAT reagent labels cysteines, never goes to completion, according to Righetti, even if one allows the reactions to proceed overnight. Therefore, Righetti and his team searched for the optimal alkylating agent that could label all the cysteines of a pro-
A N A LY T I C A L C H E M I S T R Y / A P R I L 1 , 2 0 0 4
tein in solutions that are typically used for 2-D mapping. “We tried just about everything,” he says. Finally, the researchers stumbled upon two compounds that fit the bill: 2- and 4-vinylpyridine (2VP and 4VP). To test the utility of these chemicals for proteomics, Righetti’s group challenged them with various protein substrates. 4VP successfully labeled chicken egg lysozyme, despite the presence of many positive charges on its surface. Righetti says that 4VP “can navigate among the positive charges and be attracted by the negative charges of the –SH groups of the cysteines—that’s what we think.” The researchers used MALDI-TOF MS to monitor the alkylation of higher mass proteins, such as human serum albumin and rabbit phosphorylase, by both 2VP and 4VP. They compared the results with those obtained with a different alkylating agent, iodoacetamide. Whereas iodoacetamide alkylated ~ 80% of the cysteines in each protein after 6 h, the VP reactions went to completion within 1 h. Finally, VP was tested for use in quantitative proteomics studies. -Lactalbumin was labeled in two separate reactions by light (non-deuterated) or heavy (deuterated) 2VP. The protein was then digested, and the peptides were analyzed by MS. Cysteine-containing peptides from heavy -lactalbumin had higher m/z values than those originating from -lactalbumin treated with light 2VP, which indicates that the cysteines were labeled. Identical results were obtained from mixtures of labeled peptides. The researchers are now using this new labeling method in cancer studies. According to Righetti, curing cancer is what it is all about. “We all look for the golden protein or groups of proteins that you will inhibit with some drug and [that] will save you from tumors,” he says. “So far, it’s very hard, but we keep digging.”
