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Optimization of methods for the quantitative analysis of global cell surface proteome and cell surface polarization

ABSTRACT: The application of our cell surface labeling and peptide enrichment workflow to MDCKII epithelial monolayers enabled large-scale quantification of apical versus basolateral domains, yielding over 2,100 proteins with 235 showing significant polarized distribution in agreement with known biology. This method offers high specificity for extracellular labeling and quantitative resolution of cell surface protein polarization, providing a powerful platform for studying epithelial biology and identifying extracellular epitopes relevant to diagnostics and therapeutic targeting.

ORGANISM(S): Canis

SUBMITTER: Zoltan Szabo

PROVIDER: PXD071272 | JPOST Repository | Thu Nov 27 00:00:00 GMT 2025

REPOSITORIES: jPOST

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			Action	DRS
	Peptide-1-1-Apical.raw	Raw
	Peptide-1-1-Baso.raw	Raw
	Peptide-1-2-Apical.raw	Raw
	Peptide-1-2-Baso.raw	Raw
	Peptide-2-1-Apical.raw	Raw

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Optimization of methods for the quantitative analysis of global cell surface proteome and cell surface polarization

Project description:he application of our cell surface labeling and peptide enrichment workflow to MDCKII epithelial monolayers enabled large-scale quantification of apical versus basolateral domains, yielding over 2,100 proteins with 235 showing significant polarized distribution in agreement with known biology. This method offers high specificity for extracellular labeling and quantitative resolution of cell surface protein polarization, providing a powerful platform for studying epithelial biology and identifying extracellular epitopes relevant to diagnostics and therapeutic targeting.

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Rapid enzyme-mediated biotinylation for cell surface proteome profiling

Project description:Cell surface is the primary site for sensing the extracellular stimuli. The knowledge of the transient changes on surfaceome upon a perturbation is very important as the initial changed proteins could be driving molecules for some phenotype. However, one or multi-step long-time handling procedures are typically required for efficient labeling by current cell-surface labeling strategies partly due to their slow labeling kinetics. In this study, we developed a fast cell-surface labeling strategy, termed as peroxidase-mediated cell-surface labeling (PECSL), enabling efficient and selective labeling of cell-surface proteins within seconds. With a labeling time of 1 min, 2684 proteins, including 1370 cell surface-annotated proteins (cell surface/plasma membrane/extracellular), 734 transmembrane proteins, and 81 CD antigens, were identified by PECSL technology from HeLa cells. By comparison with negative control experiment using quantitative proteomics, the cytoplasmic contaminations from avidin-affinity purification could be reduced and 500 out of the 731 significantly enriched proteins in positive experimental groups (p-value < 0.05, ≥ 2-fold) were cell surface-annotated proteins, including 393 (54%) plasma membrane proteins. Finally, this technology was applied to track the dynamic changes of surfaceomes upon insulin stimulation at two-time points (5 min and 2 h) in HepG2 cells. 32 proteins were found to be significantly regulated by different regulatory mechanisms, including many known insulin-related proteins, like INSR, CTNNB1, TFRC, IGF2R, SORT1. We envision that this technique could be a powerful tool to analyze the transient changes of surfaceomes with a good time resolution and delineate the temporal and spatial regulation of cellular signaling.

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Natural display of nuclear-encoded RNA on the cell surface and its impact on cell interaction [Surface-FISHSeq]

Project description:Compared to proteins, glycans, and lipids, much less is known about RNAs on the cell surface. We developed a series of technologies to test for any nuclear-encoded RNAs that are stably attached to the cell surface and exposed to the extracellular space, hereafter called membrane-associated extracellular RNAs (maxRNAs). We visualized maxRNAs on the cell surface, sequenced selected maxRNAs, and validated two maxRNAs by RNA fluorescence in situ hybridization. To test for cell-type specificity of maxRNA, we used randomized antisense oligos to hybridize to single-stranded transcripts exposed on the surface of human peripheral blood mononuclear cells (PBMCs). Combining this strategy with imaging flow cytometry, single-cell RNA sequencing, and maxRNA sequencing, we identified monocyte as the major type of maxRNA+ PBMCs and prioritized 11 maxRNAs for functional tests. Extracellular hybridization of antisense oligos to the cell surface-exposed fragments of FNDC3B and CTSS transcripts inhibited monocyte adhesion to vascular endothelial cells. Collectively, these data highlight maxRNAs as a functional component of the cell surface, suggesting an expanded role for RNA in cell-cell interaction and communication.

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Project description:Shuster SA, Li J, Chon U, Hu MC, Luginbuhl DJ, Udeshi ND, Carey DK, Takeo YH, Xu C, Mani D.R., Han S, Ting AY, Carr SA, Luo L. Cell-surface proteins mediate cell-cell interactions throughout the lifetime of multicellular organisms, yet there are no general methods for profiling them in vertebrate tissues. Here, we present in-situ cell-surface proteome extraction by extracellular labeling (iPEEL) in mice, which enables cell-type-specific profiling of cell-surface proteomes in native tissues. Applying iPEEL to developing and mature cerebellar Purkinje cells revealed a differential enrichment in cell-surface proteins with post-translational processing and synaptic functions in the developing and mature proteomes, respectively. A proteome-instructed in vivo loss-of-function screen identified a critical role for Armadillo-like helical domain-containing protein 4 (Armh4) in Purkinje cell dendrite morphogenesis. Armh4 overexpression also disrupts dendrite morphogenesis; this effect requires its conserved cytoplasmic domain and is augmented by disruption of endocytosis. Our results highlight the utility of cell-surface proteomic profiling in native tissues in identifying regulators of cell-surface signaling events.

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