Bottom Up Mass Spectrometry Analysis of the PD-L1 Overexpressed Cell Proteome

PD-L1 is a clinically relevant immune checkpoint. It is expressed on the membranes of somatic cells and deactivates immune cells that express PD-1. Much effort has been given to understanding how PD-L1 expression and stability is regulated. In the present study, we generated cell lines overexpressing PD-L1 using the bone cancer cell line, U2OS. This was accomplished by inserting a modified PD-L1 gene tagged with a GFP domain. Control cells did not receive the transgene. Cell lysates were collected from control and PD-L1 overexpressing cells and prepared for mass spectrometry analysis using the SPEED protocol. Mass spectrometry analysis on the samples was conducted using a bottom-up proteomics approach on a Thermo Scientific Orbitrap Q Exactive Plus LC-MS instrument. The resulting mass spectra were processed using the Proteome Discoverer software to detect PD-L1 overexpression, changes in PD-L1 regulation, as well as ubiquitination state. The proteome analysis revealed consistently high levels of PD-L1 across all experimental samples, highlighting its potential role in the studied biological context. These findings demonstrate the efficacy of mass spectrometry-based proteomics for detecting PD-L1 expression and post-translational modifications, contributing to a deeper understanding of its regulation.