Dataset Information

Global surface proteomes of hPSC-derived brain microvascular endothelial-like cells (hPSC-BMELCs), human cerebral microvascular endothelial cell line D3 (HCMEC/D3), and human umbilical vein endothelial cells (HUVECs)

ABSTRACT: Abundance proteomics analysis was performed to characterize the surface proteome of three cell lines: hPSC-BMELCs, a stem cell derived model for human blood-brain barrier endothelial cells; HCMEC/D3, an immortalized cell line of brain microvascular endothelial cells; and HUVECs, primary human endothelial cells isolated from the umbilical vein. The Thermo Scientific Pierce Cell Surface Protein Biotinylation and Isolation Kit was used which enables biotinylation and isolation of cell surface proteins by labeling with EZ-Link Sulfo-NHS-SS-Biotin, a thiol-cleavable amine-reactive biotinylation reagent, followed by capture with NeutrAvidin Agarose and reduction of the disulfide bond with DTT for protein release. Technical replicates of cells treated with biotin and without biotin were included for each cell line.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Endothelial Cell Of Umbilical Vein, Brain Microvascular Endothelial Cell, Endothelial Blood Brain Barrier

SUBMITTER: Mehdi Bouhaddou

LAB HEAD: Mehdi Bouhaddou

PROVIDER: PXD059899 | Pride | 2025-11-09

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	PA2_table_of_crossrun_normalized_feature_levels.csv	Csv
	PA2_table_of_crossrun_normalized_protein_levels.csv	Csv
	PA2_table_of_quantitative_comparisons.csv	Csv
	checksum.txt	Txt
	metadata.txt	Txt

Items per page:

1 - 5 of 7

Similar Datasets

Project 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.

Dataset Information

Global surface proteomes of hPSC-derived brain microvascular endothelial-like cells (hPSC-BMELCs), human cerebral microvascular endothelial cell line D3 (HCMEC/D3), and human umbilical vein endothelial cells (HUVECs)

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets