Proteomics

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Rapid enzyme-mediated biotinylation for cell surface proteome profiling


ABSTRACT: 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.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Yanan Li 

PROVIDER: PXD022744 | JPOST Repository | Fri Nov 26 00:00:00 GMT 2021

REPOSITORIES: jPOST

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