| Added by | mollevi |
|---|---|
| Last modified by | jacques.colinge |
| Group name | EquipeJC |
| Item Type | Journal Article |
| Title | A surface biotinylation strategy for reproducible plasma membrane protein purification and tracking of genetic and drug-induced alterations |
| Creator | Hormann et al. |
| Author | K. Hormann |
| Author | A. Stukalov |
| Author | A. C. Muller |
| Author | L. X. Heinz |
| Author | G. Superti-Furga |
| Author | J. Colinge |
| Author | K. L. Bennett |
| Abstract | Plasma membrane (PM) proteins contribute to the identity of a cell, mediate contact and communication and account for more than two thirds of known drug targets1-8. In the past years several protocols for the proteomic profiling of PM proteins have been described. Nevertheless, comparative analyses have mainly focused on different variations of one approach9-11. We compared sulfo-NHS-SS-biotinylation, aminooxy-biotinylation and surface coating with silica beads to isolate PM proteins for subsequent analysis by one-dimensional gel-free liquid chromatography mass spectrometry. Absolute and relative numbers of PM proteins and reproducibility parameters on a qualitative and quantitative level were assessed. Sulfo-NHS-SS-biotinylation outperformed aminooxy-biotinylation and surface coating using silica beads for most of the monitored criteria. We further simplified this procedure by a competitive biotin elution strategy achieving an average PM annotated protein fraction of 54 % (347 proteins). Computational analysis using additional databases and prediction tools revealed that in total over 90 % of the purified proteins were associated with the PM, mostly as interactors. The modified sulfo-NHS-SS-biotinylation protocol was validated by tracking changes in the plasma membrane proteome composition induced by genetic alteration and drug treatment. GPI-anchored proteins were depleted in PM purifications from cells deficient in the GPI transamidase component PIGS; and treatment of cells with tunicamycin significantly reduced the abundance of N-glycoproteins in surface purifications. |
| Publication | J Proteome Res |
| Date | Dec 24 2015 |
| Journal Abbr | Journal of proteome research |
| DOI | 10.1021/acs.jproteome.5b01066 |
| ISSN | 1535-3907 (Electronic) 1535-3893 (Linking) |
| Tags | corresponding, original, phd, postdoc |
| Date Added | 2018/11/14 - 11:48:35 |
| Date Modified | 2019/10/22 - 21:17:24 |
| Notes and Attachments | (Note) (Note) 26699813 (Attachment) |
Institut de Recherche en Cancérologie de
