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Related Concept Videos

Proteomics01:33

Proteomics

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A proteome is the entire set of proteins that a cell type produces. We can study proteomes using the knowledge of genomes because genes code for mRNAs, and the mRNAs encode proteins. Although mRNA analysis is a step in the right direction, not all mRNAs are translated into proteins.
Proteomics is the study of proteomes' function. It involves the large-scale systematic study of the proteome to denote the protein complement expressed by a genome. Scientist Mark Wilkins coined the term...
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Related Experiment Video

Updated: Jul 5, 2025

"Cell Surface Capture" Workflow for Label-Free Quantification of the Cell Surface Proteome
06:31

"Cell Surface Capture" Workflow for Label-Free Quantification of the Cell Surface Proteome

Published on: March 24, 2023

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Magnetic Bead-Based Workflow for Sensitive and Streamlined Cell Surface Proteomics.

Dylan Z Dieters-Castator1, Paolo Manzanillo2, Han-Yin Yang1

  • 1Discovery Proteomics, Amgen Research, South San Francisco, California 94080, United States.

Journal of Proteome Research
|January 16, 2024
PubMed
Summary
This summary is machine-generated.

We optimized a magnetic bead-based Cell-Surface Capture (CSC) workflow to efficiently identify cell surface proteins, revealing significant differences between in vitro and in vivo samples for drug development.

Keywords:
N-glycopeptide enrichmentcell surface capturemass spectrometryplasma membrane N-glycoproteinsurfaceomics

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Area of Science:

  • Proteomics
  • Cell Biology
  • Biochemistry

Background:

  • Cell surface proteins are crucial for therapeutic targeting and cellular phenotyping.
  • Challenges in mass spectrometry-based proteomics include low abundance, PTMs, and hydrophobicity of surface proteins.
  • Existing methods require optimization for efficient enrichment and detection.

Purpose of the Study:

  • To optimize a Cell-Surface Capture (CSC) workflow using magnetic bead-based processing for improved identification of cell surface proteins.
  • To evaluate various parameters including labeling conditions, enrichment specificity, lysis buffers, digestion, deglycosylation, and data acquisition methods.
  • To enhance the efficiency and reproducibility of surfaceome analysis.

Main Methods:

  • Optimized a Cell-Surface Capture (CSC) workflow incorporating magnetic bead-based processing.
  • Evaluated labeling conditions (biotin tags, catalysts), enrichment (streptavidin beads), lysis buffers, digestion, deglycosylation, and data acquisition (DDA, DIA, TMT).
  • Employed semiautomated processing for sample handling and utilized single-pot solid-phased-enhanced sample-preparation (SP3).

Main Results:

  • Identified ∼600–900 cell surface N-glycoproteins from 25–200 μg of HeLa protein using the optimized CSC workflow.
  • Maximal surfaceome coverage was achieved with alkoxyamine-PEG4-biotin, SDS/urea lysis buffers, SP3, and streptavidin magnetic beads.
  • Revealed significant differences between in vitro monolayer cultures and in vivo tumor xenografts of CT26 colon adenocarcinoma.

Conclusions:

  • The magnetic bead-based CSC workflow significantly improves the efficiency and reproducibility of surfaceome analysis.
  • This optimized workflow enables identification of novel N-glycosites with reduced sample material.
  • The findings provide insights into cellular phenotypes and aid in target identification for drug development.