Acute myeloid leukaemia (AML) remains a major therapeutic challenge, largely because of its genetic heterogeneity and limited immunotherapy success beyond monoclonal anti-CD33-antibody. Recent studies have shown that AML subpopulations differ markedly in their cell-surface antigen composition, underscoring the need for comprehensive, quantitative surfaceome profiling to identify safer and more selective immunotherapeutic targets (Köhnke et al., 2022; PMID: 36574542; Bordeleau et al., 2024; PMID: 38656966). Cell-surface proteins—owing to their accessibility and central roles in signalling and immune interaction—represent a rich source of potential therapeutic entry points, yet their low abundance and the biochemical complexity of the plasma membrane present persistent analytical challenges.
To address these issues, we developed and benchmarked an optimised 96-well streptavidin-based enrichment platform that streamlines the isolation of biotinylated surface proteins, improving throughput and reproducibility while maintaining the high specificity of biotin–streptavidin capture. This refined system allows efficient processing of limited clinical input material and is adaptable for large-scale, high-throughput surfaceome profiling of patient samples. Ongoing refinements are focused on reducing cytosolic contamination—a frequent limitation in conventional workflows—to enhance purity and ensure robust surface annotation.
Using this improved platform, we compared two complementary chemoproteomic approaches: Cell Surface Biotinylation (CSB) and Cell Surface Capture (CSC), benchmarked on human AML cell lines MOLM13 and MV4-11. The CSB workflow employs amine-reactive Sulfo-NHS-LC-LC-biotin to label accessible lysines, providing a technically straightforward and high-yield approach. In contrast, CSC leverages mild periodate oxidation of terminal sialic acids followed by aminooxy-biotin coupling, specifically enriching for N-glycoproteins and enabling the detection of glycan-linked surface peptides.
Data acquisition on the Orbitrap Astral mass spectrometer using data-independent acquisition (DIA-MS) achieved deep, quantitative surfaceome coverage (~2000 putative cell surface proteins) from limited cell inputs (~4 million cells/replicate). Comparative analyses between CSB and CSC datasets demonstrated strong concordance (Pearson r > 0.85), confirming that both methods detect biologically relevant differential expression patterns between p53 knockout (p53KO) and wild-type (WT) AML cells. Canonical AML surface markers, including CD123, CLEC12A, CD96, CD117, CD33, CD47, TIM-3 and FLT3, were consistently identified across both workflows.
Together, these results validate CSC and CSB as complementary, high-fidelity strategies for AML surfaceome profiling. The scalable 96-well streptavidin platform, coupled with the sensitivity of Orbitrap Astral DIA-MS, establishes a robust foundation for high-throughput clinical surface proteomics and next-generation immunotherapeutic target discovery.