Session: Parallel session 1 - Health and biological sciences

From bulk to single cell proteomics to evaluate the effects of CeLMODs on Multiple Myeloma

Charline KELLER¹, Pedro LOPEZ-NAVARRO², Pauline PERDU-ALLOY¹, Jeewan BABU-RIJAL¹, Alexandre DETAPPE², Christine CARAPITO¹

¹Laboratoire de Spectrométrie de Masse BioOrganique, Institut Pluridisciplinaire Hubert Curien (UMR 7178), CNRS, Université de Strasbourg , Strasbourg, France
²Nanotranslational Lab, Institut de Cancérologie de Strasbourg Europe (ICANS), Strasbourg , France

Introduction

Complex cellular systems such as cancers, as Multiple Myeloma, present a wide cellular heterogeneity. CeLMODs are heterobifunctional degrading molecules binding a protein of interest, here GSPT1, to induce its degradation. Conventional bulk proteomics involve analyzing peptides mixtures from hundreds of thousands cells, making it impossible to differentiate a healthy from a disease cell. The recent introduction of Single-Cell Proteomics (SCP) is highly expected to overcome bulk limitations. Analyzing single cell proteome contents remains a major analytical challenge due to the extremely low abundance of available protein material. The development of automated sample preparation methods as well as the implementation of trapped ion mobility spectrometry coupled to a highly sensitive and ultrafast mass spectrometer (TimsQTOF) reveals to be crucial to succeed in SCP with high proteome coverage and throughput.

Methodology

CeLMODs effects on GSPT1 were evaluated on Myeloma cell lines. Myeloma cells were sorted and digested using a CellenONE robot cell sorter (Cellenion®). Peptides were separated on an Aurora Rapid CSI (IonOptiks) C18-RP (75µmx50mm,1.7µm) column using a NanoElute 2 coupled to a TimsTOF Ultra (Bruker Daltonics), using a gradient from 5% to 35% ACN in 0.1% FA over 10 min at 0.25 µL/min. Sample-specific DIA-PASEF methods were developed.

Results

Proteome coverage exceeded 8000 proteins in bulk.GSPT1 was quantified with over 30 peptides using customized DIA-PASEF. Its downregulation by the CeLMODs has been characterized, demonstrating its specific degradation upon treatments. Promising results have been obtained on single myeloma cells, reaching a proteome coverage of around 2000 proteins on cells isolated from various cell lines.

Conclusion

GSPT1 downregulation upon CeLMODs treatment has been finely characterized in bulk proteomics. Following these very promising results, the cellular drug effects on single isolated cells is currently ongoing.