Use of CE-IM-MS as a complementary tool to microfluidic-LC-IM-MS for cell surface antigen discovery

Disease 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. In this study, the use of a neutral-coated capillary was considered in order to increase peak capacity leading to a higher number of identified entities compared to uncoated capillaries. Besides, different injected sample volumes were tested using dynamic pH junction to tackle the poor sample injection volume in CE without compromising separation efficiency.
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. 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 identified by LC. Moreover, the nature of uniquely identified peptides and orthogonality of both systems were investigated.
The capability of ion-mobility (IM) was also exploited in this study. Indeed, the addition of IM 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 IM in proteomic studies to date. In this study the combination of CE with IM 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.