PXD068422

PXD068422 is an original dataset announced via ProteomeXchange.

Title	Improved T cell surfaceomics by depleting intracellularly labelled dead cells
Description	Although the plasma membrane (PM) is among the most biologically important and therapeutically targeted cellular compartments, it is among the most challenging to faithfully capture using proteomic approaches. The quality of quantitative surfaceomics data depends heavily on the effectiveness of the cell surface enrichment used during sample preparation. Enrichment improves sensitivity for low abundance PM proteins and ensures that the changes detected reflect PM expression changes rather than whole cell changes. Cell surface biotinylation with PM-impermeable, amine-reactive reagents is a facile, accessible, and unbiased approach to enrich PM proteins. For unclear reasons however, it results in unexpectedly high contamination with intracellular proteins, reducing its utility. We report that biotinylating human cells with amine-reactive reagents intracellularly labels a small but reproducible population of non-viable cells. Although these dead cells represent only 52 % of the total, we find that in T cell preparations the dead cells account for 90 % of labelled proteins. Depleting Annexin V positive dead T cells post-labelling removes ~99% of the intracellularly labelled cells, resulting in markedly improved PM identifications, peptide counts, and iBAQ intensities. Correspondingly, we found substantial depletion of intracellular proteins, particular of nuclear origin. Finally, we demonstrate that immature ER/Golgi glycoforms of CD11a and CD18 are selectively removed by dead-cell depletion. We conclude that intracellular labelling of non-viable cells is the major source of intracellular protein contaminants and can be reduced by dead-cell depletion post-labelling, improving both sensitivity and accuracy of PM proteomics.
HostingRepository	PRIDE
AnnounceDate	2026-02-09
AnnouncementXML	Submission_2026-02-08_16:18:27.318.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Haik Mkhikian
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: NEWT:9606;
ModificationList	monohydroxylated residue
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2025-09-16 11:47:57	ID requested
⏵ 1	2026-02-08 16:18:29	announced

S, á, nchez CD, Balakrishnan A, Krisko B, Ahmmed B, Witchey L, Valenzuela O, Minasyan M, Pak A, Mkhikian H, Improved T Cell Surfaceomics by Depleting Intracellularly Labelled Dead Cells. Mol Cell Proteomics, 25(2):101503(2026) [pubmed]

10.1016/j.mcpro.2025.101503;

submitter keyword: glycosylation, intracellular pool,Surfaceome, biotinylation, plasma membrane

Haik Mkhikian
contact affiliation	Department of Pathology and Laboratory Medicine, School of Medicine, University of california Irvine.
contact email	hmkhikia@uci.edu
lab head
Haik Mkhikian
contact affiliation	Department of Pathology and Laboratory Medicine, School of Medicine, University of California Irvine.
contact email	hmkhikia@uci.edu
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD068422
     1. Label: PRIDE project
     2. Name: Improved T cell surfaceomics by depleting intracellularly labelled dead cells