Cell surface proteomics of 3D cell culture models: optimization, comparison, and critical evaluation of different enrichment strategies

Cell surface proteins (CSPs) represent invaluable resources in biomedical research. Located at the interface between the cell and its environment, they orchestrate numerous physio-pathological mechanisms and constitute prime candidates for therapeutic targets and diagnostic development. Yet, obtaining a comprehensive and unbiased characterization of the surfacome remains challenging. Recent analytical innovations have substantially improved our capacity to explore the surfacome using targeted strategies. Nevertheless, some of the most common strategies nowadays for untargeted CSP enrichment and high-resolution mass spectrometry (HR-MS) analysis are biotinylation-based affinity purification and plasma membrane fractionation [1]. However, the growing diversity of chemistries and workflows has also highlighted a major finding: different extraction and labelling strategies yield different protein identification profiles, and no strategy is able to give a comprehensive one-shot picture of the surfacome. Selecting the most suitable approach is therefore critical to develop a time- and cost-effective high- throughput workflow able to be scaled-up to larger sample numbers and sources and offer the most comprehensive surfacome characterization possible.
This study is part of a broader project aiming to decipher the surfacome of glioblastoma (GBM), the most widespread brain tumor in adults, with an annual incidence of over 3 per 100,000 individuals and a median age at diagnosis of 65 years. GBM’s median survival is only a few months, and its resistance to current state-of-the-art care treatments underscores the urgent need for new therapeutic strategies and therapeutic targets identification [2].
In this context, the best strategy for the extraction of CSP from GBM stem-like cell (GSC) three- dimensional culture models was evaluated by comparing different biotinylation-based enrichment strategies targeting glycans on glycoproteins, primary amine groups on lysins, and N-terminal moieties, or carboxyl groups on aspartic acid, glutamic acid, and C-terminal moieties. Following biotin-based enrichment, the protein profiles obtained from these strategies were compared with those derived from membrane precipitation workflows and whole-cell lysates, allowing us to evaluate differences in proteome coverage in terms of nature and number of identifications. This cross-comparison revealed differences in the extracted protein repertoires, highlighting the method-dependence of CSP identification and the risk of drawing incomplete conclusions when relying on a single enrichment approach.
Overall, this study allowed us to select the most effective strategy for CSP extraction from GSCs and demonstrates the importance of tailoring analytical workflows to specific clinical contexts and research objectives. Beyond its immediate application to GBM study, this work reinforces the critical role of advanced analytical techniques in refining our understanding of the surfacome and accelerating the discovery of clinically relevant biomarkers and therapeutic targets.
References:
1. A. Laurent, A. Allard, M.Fillet. TrAC. 184 (March 2025), 118143.
2. C. McKinnon, M. Nandhabalan, S.A. Murray, P. Plaha. BMJ 2021, n1560.