Background Immuno-oncology (IO) has substantially improved the survival of cancer patients over the past several years encouraging the discovery of novel IO targets which are typically proteins expressed on the surface of immune cells. Sensitive quantification of proteins in complex biological samples is routinely achieved by immunoassays that use antibodies specific to target proteins. Such approaches can be a limitation in IO drug discovery since the development of de novo antibodies is associated with long lead times, high costs, and high failure rates.

The main purpose of this work is to test whether it is possible to substitute antibody based validation for mass spectrometry (MS) based since protein quantification using MS is agnostic to species and matrices and removes the barriers of availability or specificity of antibody-based assays.

Methods For parallel reaction monitoring (PRM)-MS, proteins were extracted from cells, denatured, digested with trypsin prior to LC-MS analysis.

Results We first tested the accuracy of the PRM targeted MS True Signature™ (PRM-MS) advanced proprietary proteomics workflow developed by Biognosys by comparing the quantification of a multiplexed panel of known B cells surface antigens in hematopoietic cell lines using TrueSignature™ with the gold-standard QIFI® measurement by flow cytometry. We observed a strong correlation (r²=0.7534) between the quantitative data derived from our MS based proteomics workflow and the gold-standard QIFI® measurement, demonstrating the accuracy of the MS based method for protein quantification.

We then designed two multiplexed panels of known and novel targets in activated T cells or in Macrophages and quantified their expression using targeted MS. We could use the relationship established between the MS and QIFI® measurement to interpolate the values obtained by MS to values in molecules per cell, a typical unit for the absolute quantification of therapeutic targets.

Conclusions In conclusion, we observed a strong correlation between the quantitative data derived from our TrueSignature™ MS-based proteomics workflow with the gold-standard QIFI® measurement by flow cytometry, supporting the possibility to substitute immunoassays by mass spectrometry for the targeted multiplexed quantification of proteins. This opens new avenues in the way we can apply mass spectrometry to our research since it becomes an attractive alternative to antibody based assays. It will enable to reduce workload, increase speed, address problem of antibody availability or specificity, and provide an absolute quantification of novel IO targets.