Profiling Multiplexed Protein-Specific Sialylation in Cancer Drug Resistance Using Proximity Ligation Sequencing.

Aberrant glycosylation, particularly enhanced sialylation of membrane proteins, acts as a critical regulator of receptor signaling, immune evasion, and therapeutic resistance in cancer. However, systematic and multiplexed interrogation of protein-specific sialylation in living cells remains technically challenging. Here, we report SATP-seq, a next-generation sequencing-based strategy that integrates a sialic acid-reactive probe (SA) with a targeted protein probe (TP) to enable ensemble profiling of protein-specific sialylation. By combining metabolic glycan labeling with nanobody- or aptamer-mediated protein recognition, SATP-seq achieves dual recognition of sialic acids and protein epitopes through DNA-programmed proximity ligation. The resulting ligation products encode sialylation states into unique DNA barcodes, converting glycosylation information into sequencable signals for multiplexed and quantitative analysis within a single sequencing run. Application of SATP-seq to gefitinib-sensitive PC9 and gefitinib-resistant PC9GR cells enables parallel profiling of seven membrane glycoproteins and reveals resistance-associated remodeling of protein-specific sialylation. Notably, differential sialylation of EGFR and CD47 emerges between sensitive and resistant cells, suggesting coordinated reprogramming of proliferative and immune-regulatory pathways. Enzymatic desialylation, EGFR knockdown, and proteomic validation collectively confirm the specificity and biological relevance of these alterations. Together, these findings establish multiplexed protein-specific sialylation profiling as a scalable approach for dissecting glycosylation-driven mechanisms of cancer drug resistance.