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RESEARCH PROFILES
Shaving proteins off the plasma membrane
PLD, to obtain the enzyme. In human cells, GPI-APs are typical ly found in detergent-resistant mem branes, or lipid rafts. So to obtain GPIAPs, the researchers isolated lipid rafts from HeLa cells by using a sucrose gra dient. To harvest GPI-APs from plant cells, Jensen and colleagues used Arabidopsis thaliana plasma membrane preparations. After the lipid raft or plasma mem brane samples are prepared, a deter gent is added. The aqueous and de tergent phases are mixed together in
Detergent Aqueous
pure GPI-AP was identified and se quenced, will be published in a sepa In most proteomics studies, cells are rate paper. lysed and then the proteins inside are Until the researchers devise a way to perform the hydrophilic-interac identified and analyzed. But Ole N. Jen sen at the University of Southern Den tion chromatography analysis on a pro teomics scale, they must verify GPImark (USD) says that a lot of informa tion can be gained by studying the APs computationally. The sequences of proteins that are attached to the outer the identified proteins were analyzed surface of a cell. Proteins that are “ex by three independent programs for the posed on the surface of the cell will tell presence of GPI anchor sequence mo you what is going on with the cell,” he tifs. Proteins were considered to be true GPI-APs if more than one program explains. As described in this issue of JPR identified them as modified proteins. (pp 935–943), Jensen and col Of the 42 human proteins that leagues at USD, the John Innes were initially identified, 11 were validated as true GPI-APs. Centre (U.K.), and the Uni versity of Berne (Switzerland) In the plant preparation, 35 have taken a “shave-and-con of 42 proteins were validated as GPI-APs. According to Jen quer” approach to the study of a subset of plasma mem sen, the proteins that were not validated as GPI-APs probably brane proteins called glyco Plasma Cells GPI-AP GPI-AP Bioinformatic sylphosphatidylinositol-an leaked into the aqueous phase membrane isolation identification analysis extraction chored proteins (GPI-APs), during the recovery step. “You which are involved in many never get a completely pure Shave and conquer. Schematic of the method used to isolate and important cellular process preparation, no matter what characterize GPI-APs from human and plant cells. es. In the method, an enzyme type of proteomics [experi called GPI-specific phospho ment] you do,” he points out. In addition to several expected GPIlipase D (PLD) acts like a razor to slice a suspension at 37 °C, and PLD is add APs, a few GPI-APs that had never been GPI-APs from cell surfaces at the GPI ed to cleave the GPI-APs from the lipid tether. The proteins are then recovered membranes. After 2 additional temper detected experimentally were identi in a two-phase separation and analyzed ature shifts, the aqueous and detergent fied in the study. Interestingly, sever al GPI-APs were newly identified be by MS. The researchers identified more phases separate and then the GPI-APs GPI-APs in this study than have ever are harvested from the aqueous phase. cause of changes in the databases that been isolated in a single proteomics To identify the proteins obtained by the researchers searched. “In our ear experiment. PLD treatment, the researchers digest lier studies, we did not assign some A related enzyme, phosphatidylinosi ed these samples with trypsin and ana of the proteins as GPI proteins be tol-phospholipase C (PI-PLC), has been lyzed the peptides by LC/MS/MS. cause the computer tools didn’t ac used as the enzyme razor for several When a phospholipase cleaves the cept them based on the sequence, but previous proteomics studies. Although GPI anchor, it leaves behind a tag on the the sequences have been changed in PI-PLC is specific for GPI anchors, it C-terminus of the protein. Jensen says the meantime,” says Jensen. “The open does not cleave well when a GPI an that the presence of this tag on one of reading frame has been redefined,” he the peptides of a GPI-AP would be proof explains. chor is decorated with sugars and lip ids. “Based on the literature, it looked that the identified protein was actual The researchers are still improving like [PLD] would accept a more varied ly modified with an anchor. However, the membrane preparation methods so that they can isolate additional GPIsubstrate,” says Jensen. A more gener none of the peptides recovered by the al enzyme, he reasoned, could enable researchers had this tag. Jensen sus APs. And once they have better meth ods in hand, they plan to use GPI-APs researchers to identify many addition pects that these hydrophilic glycopep al GPI-APs. According to Jensen, this tide tags were lost in the LC step of the as markers to study diseased cells and study is the first in which PLD has been analysis. “We have shown now that we stem cells, says Jensen. He adds that in reported as a tool for GPI-AP analysis. can recover [these peptides] using hy the future, vaccines and drugs could be developed to target the GPI-APs that are In fact, PLD is not even commercially drophilic interaction chromatogra available yet, so he teamed up with Urs phy,” he says. The results of that study, identified by proteomics studies. Brodbeck, whose group was studying in which the tagged peptide from one —Katie Cottingham
738 Journal of Proteome Research • Vol. 5, No. 4, 2006
© 2006 American Chemical Society
