Proteomics

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A surface biotinylation strategy for reproducible plasma membrane protein purification and tracking of genetic and drug-induced alterations


ABSTRACT: In the past years several protocols for the proteomic profiling of plasma membrane proteins have been described. Nevertheless, comparative analyses have mainly focused on different variations of one approach [1-3]. To pave the way to high-performance differential plasma membrane proteomics, we compared sulfo-NHS-SS-biotinylation, aminooxy-biotinylation and surface coating with silica beads to isolate plasma membrane proteins for subsequent analysis by one-dimensional gel-free liquid chromatography mass spectrometry. Absolute and relative numbers of plasma membrane 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 introducing a competitive biotin elution strategy, for which the average plasma membrane annotated protein fraction amounted to 54 % (347 proteins). Moreover, purified non-plasma membrane annotated proteins and plasma membrane annotated proteins displayed similarly high reproducibility suggesting specific co-purification. In fact, the non-plasma membrane annotated data was extremely enriched for direct interactors of purified plasma membrane proteins. Computational analysis using additional databases and prediction tools revealed that in total over 90 % of the purified proteins were associated with the plasma membrane. 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 plasma membrane 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. Altogether, this study introduces an improved, filter-free sulfo-NHS-SS-biotinylation protocol and demonstrates it to be a specific, effective and reproducible method to isolate proteins associated with the plasma membrane, thus enabling future large-scale comparative cell surface mappings.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Katrin Hörmann  

LAB HEAD: Keiryn L. Bennett

PROVIDER: PXD002141 | Pride | 2022-02-22

REPOSITORIES: Pride

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A Surface Biotinylation Strategy for Reproducible Plasma Membrane Protein Purification and Tracking of Genetic and Drug-Induced Alterations.

Hörmann Katrin K   Stukalov Alexey A   Müller André C AC   Heinz Leonhard X LX   Superti-Furga Giulio G   Colinge Jacques J   Bennett Keiryn L KL  

Journal of proteome research 20160112 2


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 subs  ...[more]

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