PXD002141

PXD002141 is an original dataset announced via ProteomeXchange.

Title	A surface biotinylation strategy for reproducible plasma membrane protein purification and tracking of genetic and drug-induced alterations
Description	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.
HostingRepository	PRIDE
AnnounceDate	2024-10-22
AnnouncementXML	Submission_2024-10-22_05:33:13.312.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Katrin Hörmann
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	biotinylated residue; monohydroxylated residue; iodoacetamide derivatized residue
Instrument	LTQ Orbitrap Velos

Revision	Datetime	Status	ChangeLog Entry
0	2015-05-05 06:26:46	ID requested
1	2022-02-22 03:45:45	announced
⏵ 2	2024-10-22 05:33:14	announced	2024-10-22: Updated project metadata.

10.1021/acs.jproteome.5b01066;

H, ö, rmann K, Stukalov A, M, ü, ller AC, Heinz LX, Superti-Furga G, Colinge J, Bennett KL, A Surface Biotinylation Strategy for Reproducible Plasma Membrane Protein Purification and Tracking of Genetic and Drug-Induced Alterations. J Proteome Res, 15(2):647-58(2016) [pubmed]

curator keyword: Technical

submitter keyword: KBM7, MCP, technical paper, cell surface proteomics

Keiryn L. Bennett
contact affiliation	CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences
contact email	kbennett@cemm.oeaw.ac.at
lab head
Katrin Hörmann
contact affiliation	CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences
contact email	khoermann@cemm.oeaw.ac.at
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD002141
     1. Label: PRIDE project
     2. Name: A surface biotinylation strategy for reproducible plasma membrane protein purification and tracking of genetic and drug-induced alterations