Thorup John R, Johnston Sarah A, Haines Moe, Sedodo Edwin, O'Neill Kathleen, Drapkin Ronald, Udeshi Namrata D, Gillette Michael A, Carr Steven A, Satpathy Shankha
Broad Institute of MIT and Harvard, Cambridge, MA, USA.
Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
bioRxiv. 2025 Jul 20:2025.07.18.664488. doi: 10.1101/2025.07.18.664488.
Cell surface proteins (CSPs) regulate key cellular functions and represent valuable targets for diagnostics and therapeutics. Despite advances in proteomic workflows, CSP analysis from cryopreserved or low-input clinical samples remains limited by technical constraints, including reduced membrane integrity, inefficient labeling, and high background. To address these challenges, we optimized and benchmarked two complementary surface enrichment strategies compatible with low-input applications (fewer than 1 million cells) and real-world sample types, including fresh, viably cryopreserved, and dissociated solid tissues. We systematically compared oxidation-based N-glycoprotein capture and WGA-HRP-mediated proximity labeling across a range of input amounts using both solid tumor (A549) and hematologic cancer (KMS-12-BM) cell lines. The N-glycopeptide method yielded superior specificity in low-input contexts, while WGA-HRP captured complementary CSP subsets. Together, the methods identified more than 700 CSPs, with approximately 175 unique identifications per protocol. Both workflows detected dynamic EGFR internalization following EGF stimulation and maintained high reproducibility (Pearson correlation greater than 0.9) between fresh and cryopreserved preparations. To extend these findings to tissue-derived samples, we optimized dissociation protocols for healthy endometrium and applied the N-glycopeptide method to cryopreserved dissociated endometrium from three healthy donors. Enzymatic dissociation enabled accurate CSP profiling from fewer than 1 to 2 million cells. This study provides a systematic comparison of two leading surface proteomics approaches, validates their performance on cryopreserved and low-input specimens, and demonstrates applicability to clinically relevant tissues. Our optimized workflows enable robust surfaceome characterization in translational settings where sample quantity and preservation methods are often limiting, opening new avenues for biomarker discovery and patient stratification.
细胞表面蛋白（CSPs）调节关键的细胞功能，是诊断和治疗的重要靶点。尽管蛋白质组学工作流程取得了进展，但从冷冻保存或低输入量的临床样本中进行CSP分析仍然受到技术限制，包括膜完整性降低、标记效率低下和背景噪音高。为了应对这些挑战，我们优化并对比了两种互补的表面富集策略，它们适用于低输入量应用（少于100万个细胞）和实际样本类型，包括新鲜的、可存活冷冻保存的以及解离的实体组织。我们使用实体瘤（A549）和血液系统癌症（KMS-12-BM）细胞系，在一系列输入量下系统地比较了基于氧化的N-糖蛋白捕获和WGA-HRP介导的邻近标记。N-糖肽方法在低输入量情况下具有更高的特异性，而WGA-HRP捕获了互补的CSP子集。这两种方法共鉴定出700多种CSP，每个方案约有175个独特鉴定。两种工作流程都检测到了EGF刺激后EGFR的动态内化，并在新鲜和冷冻保存的样本之间保持了高重现性（皮尔逊相关系数大于0.9）。为了将这些发现扩展到组织来源的样本，我们优化了健康子宫内膜的解离方案，并将N-糖肽方法应用于来自三名健康供体的冷冻保存的解离子宫内膜。酶解能够从少于100万至200万个细胞中准确进行CSP分析。本研究系统比较了两种领先的表面蛋白质组学方法，验证了它们在冷冻保存和低输入量样本上的性能，并证明了其在临床相关组织中的适用性。我们优化的工作流程能够在样本数量和保存方法通常受限的转化环境中进行强大的表面组表征，为生物标志物发现和患者分层开辟了新途径。
