A Surface Biotinylation Strategy for Reproducible Plasma Membrane Protein Purification and Tracking of Genetic and Drug-Induced Alterations
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- 作者：Katrin H?rmann ; Alexey Stukalov ; André C. Müller ; Leonhard X. Heinz ; Giulio Superti-Furga ; Jacques Colinge ; Keiryn L. Bennett
- 刊名：Journal of Proteome Research
- 出版年：2016
- 出版时间：February 5, 2016
- 年：2016
- 卷：15
- 期：2
- 页码：647-658
- 全文大小：629K
- ISSN：1535-3907
文摘
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 targets.1?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 approach.9?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. Glycosylphosphatidylinositol (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.