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Sweetening glycoprotein processing
canavalin A (ConA)-agarose, which binds to glycoproteins. After the glycoprotein-enriched fraction is eluted from the column with strong glycan buffer, it is acidified and loaded onto the reactor under pressure, where it becomes concentrated on the beads. One big advantage is that the glycan buffer, which is not compatible with MS, does not need to be removed be-
reduced and then digested with trypsin while still attached to the beads. The resulting peptides are fractionated in the Reasoning that glycoproteins would be reactor through five stepwise changes in easier to find if they were concentrated pH, and the eluates are analyzed with in a small volume rather than swimLC/MS/MS. “In the past, when we have ming around in a test tube, researchers used the reactor for proteomics, we did have invented a compact glycoprojust one-step elution,” Figeys says. “We teomic reactor. In this issue of JPR thought that if we incorporated step elu(DOI 10.1021/pr800734r), Daniel Figeys tion, that level of fractionation would and colleagues at the University of Otallow us to see more proteins tawa and the Chinese Acadand glycosylated proteins.” emy of Sciences report the They found that step pH eluidentification of 41 unique tion increased the number of glycoproteins from as little as identifiable glycoproteins by 5 µL of human plasma with a ∼45%. “Both glycopeptides reactor that performs all of and nonglycosylated tryptic the processing steps between peptides are analyzed by LC/ lectin chromatography and MS/MS, resulting in higher MS. “You reduce the volume protein sequence coverage and so you increase the concenmore reliable identifications,” tration ∼1000-fold and also says first author Hu Zhou, a reduce background noise,” postdoc in Figeys’s group. Figeys says. After processing 5 µL of The glycoproteomic reachuman plasma in the reactor, tor is an offspring of a prothe researchers detected 82 teomic reactor unveiled by unique glycopeptides from 41 Figeys 3 years ago (J. Prounique proteins. “So, we teome Res. 2006, 5, identified 16 unique glyco2754-2759). “We started all peptides per µL of human this by thinking that a cell plasma, while other studies has a very small volume,” he have ranged between 2 and explains. “So the goal is to Compact chemistry. Samples enriched in glycoproteins are prepared 8,” Figeys says. try to re-create that to profor MS in the glycoproteomic reactor. As well as being efficient vide better yields.” and fast, the glycoproteomic The new reactor is designed reactor is also cheap to make to process N-linked glycoprofore a sample is loaded. and use. Its most expensive compoteins, in which the carbohydrate moiety Once the enriched fractions enter the nent is the pressurized vessel, and it is hooked to an asparagine residue. Bereactor, carbohydrate moieties are rerequires only small amounts of cause many N-linked glycoproteins are moved from N-linked glycoproteins with reagents. displayed on the outer surface of a peptide-N-glycosidase F. This amidase The group is currently making the plasma membrane or secreted from leaves aspartate in place of asparagine reactor more robust. They are also cells, they are readily accessible for cliniand labels the aspartate with 18O if heavy devising reactors for other proteins cal purposes. N-linked glycoproteins with posttranslational modifications, captured Figeys’s interest because he is water is provided. “Without the label, it such as phosphorylation and O-glycosearching for markers for Alzheimer’s is possible you could have only a mass sylation (in which the carbohydrate and Parkinson’s diseases. shift of 0.97 daltons after you cleave off moiety is attached to a serine or About the size of a laptop computer, the sugar,” Figeys says. “Because there threonine residue). “Once we have all the reactor contains a piece of capillary are other ways to get [such a small shift], that in place, we will consider the tubing inside a pressurized vessel, tagging the site with 18O makes you sure options for making sure that more along with a manifold and pump. The that the shift was due to the enzymatic people have access to the reactors,” tubing is closed at one end and packed treatment and not to other effects.” Figeys says, “either through commerunder pressure with cation-exchange With conventional methods, enzycialization or just showing people beads. A series of reagents flows over matic deglycosylation takes 4-12 hours. how to [make and use the reactors] the beads. In the reactor, it takes
