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R E S E A R C H Identifying membrane glycoproteins
P R O F I L E
tion. The results suggest that more than one lectin should be used for protein purification. “We’ve used additional Although lectins have been used for lectins as well and will probably extend many years to isolate glycoproteins, it is it beyond that,” says Wilkins. unclear what proportion of those proMembrane proteins are not the onteins is actually associated with memly proteins that are often extenbrane processes. Such knowledge sively glycosylated. Thus, the is important because membrane same approach can also be used proteins represent nearly 75% of to capture other glycosylated all biotherapeutics that are curproteins, such as those that are rently in clinical trials. secreted, says Wilkins. “GlycosyTo get a better feel for how lation is thought to influence many membrane glycoproteins both their stability as well as are captured by lectins, John Wiltheir interaction with other molkins and colleagues at the Univerecules. It gives us another set of sity of Manitoba (Canada) and potentially released products Beavis Informatics tested two difthat a cell might make that ferent lectin affinity matrixes— might be of relevance to these concanavalin A (Con A) and processes,” he says. wheat germ agglutinin (WGA). To make the approach more The results, which are reported in restrictive to membrane glycothis issue of JPR (pp 841–850), Seeing red. Unknown membrane proteins on the surface of a proteins, the researchers first perindicate that ∼30% of the glycohuman mesenchymal stem cell are stained red with an antibody. formed a physical separation of proteins captured by the two Actin filaments in the cell are stained green. the membrane. They then used a matrixes contain domains preseries of lectins to look at the dicted by bioinformatics to be overall compositions in terms of subsets The two lectins captured a wide integral membrane proteins. of compositions, says Wilkins. “We get range of proteins, in terms of both Membrane proteins represent tarclinical samples where we have very limtheir molecular weights and isoelectric gets that can be manipulated, which ited amounts of material,” he says. The points. “This is very encouraging becould lead to a better understanding of lectin columns were found to enrich the cause membrane proteins are particuhuman conditions such as rheumatoid glycosylated proteins from whole-cell larly difficult to deal with in terms of arthritis, renal transplantations, and lysates by an average of 150–200 fold. If 2-D separations,” says Wilkins. The lecviral infections, says Wilkins. “We feel the researchers actually get enough tistins also captured proteins of varying that they may be good targets to begin sue to do an isolation, then lectin affinity levels of abundance, including the very to look at these processes and ultimatechromatography can be used to get low abundance proteins and the quite ly interfere with them in a very cell-spesome understanding of the glycoprohigh abundance proteins. “You don’t cific way, without having the side effects teins and ultimately whether they are necessarily see identical subsets of of the more generic targeting pathways associated with membrane proteins in molecules with MALDI and ESI. We get that people are trying to interfere with,” those tissues. the feeling that electrospray lets us see he says. “The reality is, if you look at the almore low-abundance proteins than Membrane proteins are often heavgorithms that define whether a protein MALDI, but MALDI probably gives us a ily glycosylated, which allows them to is actually a membrane protein, debetter quantitative estimate of the levbind to lectins. Lectins are a group of pending on the program that you use, els of representation. One might exproteins that recognize carbohydrates you get very significant differences in pect that [ESI] will provide a better with high specificity. “They can actually terms of the frequency. There is still a compositional estimate,” adds Wilkins. differentiate between one change in a lot of information that can be gained by Con A and WGA also appear to be linkage in a carbohydrate,” says Wilkins. ultimately acquiring good membrane complementary. Out of a total of 132 Usually lectins do not contain compoprep, defining what the properties of unique membrane glycoproteins identinents of the carbohydrate chain that those are, and developing better algofied from 963 MS/MS spectra, only 26 of they recognize, he adds. rithms to identify the transmembrane them were captured by both lectins. To test the two lectin matrixes, the region,” says Wilkins. Seven of the proteins lacked any predicresearchers loaded whole-cell lysates —Britt E. Erickson ted or experimental functional descriponto affinity chromatography columns COURTESY OF JOHN WILKINS
containing either Con A or WGA. The captured proteins were eluted and subjected to protease digestion, and the digested proteins were separated by HPLC and identified by MS/MS using either MALDI or ESI.
© 2004 American Chemical Society
Journal of Proteome Research • Vol. 3, No. 4, 2004
691
