Interest of CE-IM-MS as a complementary tool to chromatographic-based methods for cell surface antigen discovery: proof-of-concept using human myeloma LP-1 cell line.

Abstract

peer reviewedDisease management of multiple myeloma is challenging owing to patients’ multiple relapses and resistance to standard treatments. Therefore, the introduction of innovative cell therapies such as chimeric antigen receptor T-cells (CAR-T cells) opened new horizons in the outcome of severe refractory patients affected by aggressive forms of the disease. However, one of the limitations of the expansion of those treatments is the lack of specific MM tumor-associated antigens that could be targeted by current immunotherapies. Indeed, effectiveness of current CAR-T cells treatments could be hampered due to possible antigen-evasion strategies. Therefore, the discovery of new cell surface antigens could be an interesting approach to avoid occurrence of resistance that could lead to treatment failures. For this purpose, the use of mass spectrometry (MS) proteomics-based methodologies was considered. Due to the high sample complexity, liquid chromatography (LC) is commonly used prior MS detection to maximize protein identifications. Similarly, capillary electrophoresis (CE) could be an alternative due to its ability to provide high efficiency and high throughput separation. However, CE-MS performance could be impaired by lower sensitivity due to poor design of CE-MS interface as well as low sample loading capacity. In this study, those issues were tackled by using an online preconcentration technique namely dynamic pH junction to enhance sensitivity as well as loading capacity. Indeed, different conditions were compared in order to increase loaded volume without compromising separation efficiency. Besides, neutral-coated capillary was used in order to increase peak capacity leading to a higher number of identified entities compared to uncoated capillaries. Since CE separation principle is orthogonal to the mechanism that drives separation in LC, the use of both techniques to analyze the same sample allowed the increase of overall information in terms of number of identified proteins. Indeed, the interest of combining electrophoretic to chromatographic approaches was confirmed for the identification of antigens at the surface of human myeloma LP-1 cell line. As a matter of fact, despite the high number of proteins identified using LC-MS, more than a half of the proteins identified in CE could not be detected by LC. The capability of ion-mobility (IM) was also exploited in this study. Indeed, the addition of IMS module between LC or CE and MS lead to better proteome coverage due to the additional dimension of separation. To the best of our knowledge, little attention has been paid to the potential orthogonality between CE and IMS in proteomic studies to date. In this study the combination of CE with IMS allowed the separation of isobaric and co-migrating peptides leading to the identification of a larger number of unique proteins, thus increasing the possibility of detecting new antigens. In conclusion, the proof of concept concerning the interest of CE-IM-MS for the discovery of cell surface antigens was achieved during this study