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At last, large-scale functional glycomics Pity the poor glycomics for glycans, because their Blotted researcher. DNA and prochemical properties are glycans tein chemists have PCR so similar. to amplify scarce samples; It’s a problem that established purification Nishimura has been Recovery as fluorescent and derivatization protoworking on for some derivatives cols to minimize sample time now. Previous work complexity; and a simple, from the lab produced defined genetic code to a carbohydrate affinity guide their efforts. matrix (called BlotGlyco) But what of those who that could capture exstudy glycans? Despite tracted oligosaccharides Fractionation growing recognition of via oxime-bond formathe importance of sugar tion between the sugar’s modifications to biologialdehyde/ketone moiety cal processes, researchand the matrix-bound ers have no facile way aminooxy group. Reto manipulate and work moval of the sugars from with the glycans that the matrix via transoxiTag exchange impart so much funcmization with O-subtion to cellular proteins. stituted aminooxy funcSugars are largely similar tional reagents solved chemically, making purione problem—that of Solidification fication procedures difinterchangeably funcficult and derivatization tionalizing the sugar a necessity. Yet, those chains. But the process methods that do exist are Construction of a fractionated was inefficient, with only laborious or inefficient, glycan array via sequential a 25% yield. and the modifications are transimination reactions. In this new study, application-specific. Nishimura and his team Sugar amplification remains an searched for other reactive groups that impossibility. But, in work published might yield more promising results. in AC (2008, 80, 1094–1101), ShinOn the basis of the observation that Ichiro Nishimura of Hokkaido Univerhydrazones are more susceptible to this sity (Japan) and colleagues describe a type of reaction, the authors compared glycan-tagging and -blotting procedure hydrazones’ transimination reactivity to that can be used to purify, separate, and that of oximes. As it turns out, hydraimmobilize sugar modifications from zone-to-oxime conversion was nearly endogenous proteins. The researchers quantitative, followed closely by hysuggest that this approach can form the drazone-to-hydrazone conversion. The basis for high-throughput functional result: a new high-density hydrazide glycomics. solid support, a bead the group termed The problem with complex sugar BlotGlyco H. analysis, according to Nishimura, is The team tested its matrix by profilthat unlike analysis of DNA and proing the N-glycan compositions of boteins, glycan biosynthesis is not temvine fetuin, human α1-acid glycoprotein plate-driven. Instead, it is a multistep, (AGP), and human serum glycoproteins. enzymatic process not easily replicated The glycans from these proteins were in a test tube. Furthermore, protein pustripped, coupled to BlotGlyco H, and rification methods such as HPLC and methyl esterified, and then the oligosacelectrophoresis do not generally work charides were recovered by transimina1354
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tion with an O-substituted aminooxy reagent (N-α-((aminooxy)acetyl)trypto phanylarginine methyl ester). Efficiency was monitored with MALDI TOFMS. The overall recovery efficiency was ~73%, with a coefficient of variance of 1% of the total population. Recovery efficiency was ~70% with a hydrazide compound (instead of an aminooxy reagent) and ~93% with trifluoroacetic acid. The group next tested their method on the highly complex glycan profile of carcinoembryonic antigen (CD66e), which contains 28 potential N-glycosylation sites. Other studies on similar proteins identified no more than 35 glycan structures. But, starting from 6 μg of CD66e purified from tumor sources, Nishimura’s team found 127, of which 52 were novel and highly branched or polylactosamine structures. The tag-conversion technology was next subjected to a more rigorous test. The authors made a microarray of AGP glycans by first capturing the sugars on BlotGlyco H, recovering them as fluorescent O-hydrazone derivatives, subjecting them to fractionation by HPLC, rederivatizing them with a biotin hydrazide, and finally capturing them on a streptavidin-coated surface plasmon resonance chip, where their interaction with lectins was monitored. According to Nishimura, the primary application of this technology is for the discovery of glycan-based biomarkers as both diagnostic and therapeutic targets. In his lab, he adds, “we employ this protocol for discovery research of disease-related biomarkers and the construction of glycan microarray[s].” Nishimura also says that both Oand N-glycans can be studied with this approach (a paper on the application to O-glycans “will be reported shortly,” he says) and that any functional reagent that can be made into an aminooxyfunctionalized form can be used in this method. Glycoblotting “is [the] only method
allowing large-scale clinical glycomics,” he says, because it requires very little starting material and, when combined with an automated system his team developed, takes only 3 hours to complete. “We think that the glycoblotting method is the PCR in glycobiology/glycotechnology research,” he adds. Though not yet available worldwide, he says, BlotGlyco H is commercially available in Japan through Funakoshi. a —Jeffrey M. Perkel
A new hemolymph sampling method for D. melanogaster Few organisms have been as critical to the understanding of human genetics as the fruit fly, Drosophila melanogaster. This tiny organism’s genome contains analogs to ~75% of diseasing-causing genes in humans, and the flies have been used as a genetic model organism for almost a century because they are highly fecund and adapt well to a laboratory environment. More recently, Drosophila has been enlisted in the field of neuroscience as a model for neurodegenerative disease. Certain amino acids and their derivatives can serve as neurotransmitters, and abnormalities in levels of these molecules may play a role in certain neurological disease states. The amino acids are contained in the hemolymph (essentially, the blood) of the fruit fly, but the diminutive size of an individual animal means it’s difficult to extract enough fluid to analyze by a traditional method such as HPLC. Usually, the whole bodies of several flies are homogenized to obtain the necessary sample size. Now, in work published in AC (2008, 80, 1201–1207), Scott Shippy and colleagues at the University of Illinois Chicago have developed a new sampling method for Drosophila that allows analysis of hemolymph from an individual animal. Shippy says that the work began when
David Featherstone, a collaborator and an expert in Drosophila genetics, wanted to measure the chemical changes that resulted from a specific genetic mutation known as genderblind (gb). “He had mutated this cysteine–glutamate exchange protein (xCT), and these fruit flies actually showed a very interesting phenotype in that they were bisexual fruit flies,” says Shippy. “He got a lot of publicity for that.” Featherstone needed a way to quantify the amino acid concentration absolutely, so he could understand how this genetic mutation caused this surprising phenotype. The scientists wanted to develop a method to extract just the hemolymph rather than homogenizing a whole animal, because including other tissues in the analysis can skew results. To take a sample, the researchers made a precise incision into the body of a fly, inserted a narrow tube, and drew out the hemolymph under vacuum. They then analyzed their samples by CE. “The challenge is the small volume,” says Shippy. “You have to manipulate 170 nL on average—that’s pretty small.” The researchers worried that their samples might evaporate appreciably in the time that it took to make the incision and extract the hemolymph, so they experimented with sampling under mineral oil. “The evaporation rates for even 100 nL of solution should be quite high,” says Shippy. “It took us 30 seconds to actually make the cut into the animal and then to bring our probe up to it, so we wanted to be sure that there wasn’t a significant amount of evaporation occurring.” Luckily, they found that their sample volumes did not change markedly when compared with open-air sampling. Despite the relative difficulty in obtaining samples, Shippy says it is worth it to get information on an individual level. “This adds another layer of analysis . . . because in some cases we have a very wide variance and in other cases we have a very narrow variance in the mutated
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D. melanogaster is the workhorse of genetics and neuroscience.
animals,” he says. “This is information that you can’t get at if you’re just looking at populations in the first place.” The researchers analyzed samples from both wild-type and gb mutant flies for arginine, glutamate, glycine, lysine, taurine, and glutamine. They found that average levels of taurine, glutamate, and glutamine in the mutants were significantly lower than in the wild-type animals, though levels of some amino acids varied greatly among individuals. Their results confirmed that the gb mutation affects the transport of these amino acids by the xCT protein. “Because the protein that was mutated was this exchange protein, it shows us that the glutamate levels were lowered in the hemolymph of the animals,” explains Shippy. “So there was less glutamate being transported out of the cell.” According to Shippy, the sampling method is not limited to Drosophila and could be used to analyze for many different hemolymph components. “Anything that you might think about measuring in blood—any chemical that might be related to the biology of the organism, which is essentially everything—we’d like to take a look at,” he says. Other groups have, for example, used hemolymph from Bombyx mori, the silkworm, for proteomic analysis. ( J. Proteome Res. 2006, 5, 2809–2814; 2007, 6, 3003–3010.) The possibilities are endless. a —Jennifer Griffiths
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