Enhanced cell surface enrichment and data-independent acquisition for in-depth profiling of multiple myeloma cell lines.

BACKGROUND

Multiple myeloma is a hematologic malignancy characterized by clonal plasma cell proliferation, with recent therapeutic advances focusing on immunotherapies targeting cell surface antigens. While several surface markers are well-characterized, there remains a critical need to identify additional specific targets for relapsed cases. Comprehensive surface proteome analysis is challenging due to the low abundance of surface proteins and limited cell numbers available from patient samples.

METHODS

We developed an optimized cell surface capture (CSC) protocol coupled with data-independent acquisition (DIA) mass spectrometry for comprehensive surfaceome profiling of multiple myeloma cell lines AMO1 and MM1.S. The method utilizes periodate-based oxidation of surface glycoproteins followed by biocytin hydrazide labeling and NeutrAvidin enrichment. Surface-labeled proteins were analyzed using DIA mode on an Orbitrap Eclipse Tribrid mass spectrometer with optimized parameters for low-input samples.

RESULTS

Our approach successfully identified and quantified 989 high-confidence surface proteins from minimal sample inputs (10-fold less than standard protocols), demonstrating excellent reproducibility between biological replicates (R=0.907-0.916). Comparative analysis revealed 790 shared proteins between cell lines, with 93 and 106 proteins uniquely expressed in AMO1 and MM1.S, respectively. Principal component analysis showed clear separation between cell lines (PC1=84.6% variance), highlighting distinct surface protein expression profiles. The method detected known myeloma markers including BCMA and identified additional potential therapeutic targets.

CONCLUSIONS

CSC-DIA methodology enables comprehensive surface proteome analysis from limited cell numbers, making it suitable for rare patient samples. This provides a robust platform for discovering novel surface antigens to advance personalized myeloma immunotherapy.