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

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.