Shaving proteins off the plasma membrane - American Chemical Society

Shaving proteins off the plasma membrane. In most proteomics studies, cells are lysed and then the proteins inside are identified and analyzed. But Ol...
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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