Proteomic Analysis of Cell Surface Proteins with Improved Specificity of Enrichment

Importance of the topic

The isolation of cell surface proteins is crucial for understanding receptor biology, drug targeting and biomarker discovery. Conventional biotinylation methods often label intracellular proteins, complicating downstream mass spectrometry analysis and reducing specificity for true surfaceome profiling.

Objectives and study overview

This work aimed to develop and validate a refined protocol for enriching cell surface proteins with reduced background contamination and compatibility with LC-MS. The new method was benchmarked against the standard Thermo Fisher Pierce™ Cell Surface Protein Isolation Kit across three human cell lines (HeLa, A549 and HCT-116), focusing on yield, specificity and proteomic coverage.

Methodology and instrumentation

The protocol improvements centered on the Sulfo-NHS-SS-Biotin labeling workflow and NeutrAvidin capture:

• Excess label removal instead of quenching to prevent intracellular labeling
• 50% reduction in biotin concentration to limit non-specific uptake
• 20–30% shorter incubation steps to reduce background
• Lysis buffer reformulated with a stronger detergent to solubilize multi-pass membrane proteins

Captured proteins were eluted with DTT, alkylated and digested using the Thermo Scientific™ EasyPep™ Mini MS Sample Prep Kit (Trypsin/Lys-C). Peptides were analyzed by label-free LC-MS on a Thermo Scientific™ Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™, with data processed in Proteome Discoverer™ and annotated via UniProtKB.

Main results and discussion

The optimized protocol delivered comparable or higher peptide yields (10–14 µg per 15 cm dish) while reducing intracellular contaminants threefold:

• Western blots showed strong enrichment of EGFR, CD55, CD44 and integrin αV with lower HSP90 and actin signals
• LC-MS revealed fivefold more surface proteins and fourfold fewer cytosolic proteins among the top 100 abundant identifications
• 92–95% of the top 50 most abundant proteins were confirmed plasma membrane or GPI-anchored via UniProtKB curation
• Distribution of single-span, multi-span transmembrane, GPI-anchored and extracellular matrix proteins was consistent across all cell lines

Benefits and practical applications

This improved workflow enhances surfaceome analysis by:

• Increasing specificity for membrane targets in drug discovery and immunogenic profiling
• Reducing sample complexity and boosting reproducibility in quantitative proteomics
• Enabling reliable monitoring of surface protein dynamics under various treatments

Future trends and potential uses

Potential developments include:

• Integration with targeted MS (e.g., PRM/SRM) for quantitative validation of membrane biomarkers
• Live-cell affinity labeling approaches for time-resolved surfaceome mapping
• Coupling with single-cell proteomics to explore heterogeneity in cell surface composition

Conclusion

The refined amine-biotinylation method achieves superior selectivity and MS compatibility, delivering high yields of true cell surface proteins with minimal intracellular contamination. This advancement supports more precise surfaceome profiling, accelerating research in cell biology and therapeutic target discovery.

Used instrumentation

• Thermo Scientific™ Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ Mass Spectrometer
• Thermo Scientific™ EasyPep™ Mini MS Sample Prep Kit (Trypsin/Lys-C digestion)
• NeutrAvidin agarose resin for biotinylated protein capture
• Invitrogen™ Power Blotter XL System for Western blot transfer

Reference

Benton B., Snovida S., Herting K., Zhu H., Rogers J.C., Kaboord B. Proteomic Analysis of Cell Surface Proteins with Improved Specificity of Enrichment. Thermo Fisher Scientific; Rockford, IL; 2018.