Proteasome Profiling of Non-Small Cell Lung Carcinoma

Introduction:

The mammalian proteasome is estimated to degrade ~70% of all intracellular proteins and plays a key role in antigen processing and MHC presentation. Recently we have developed a system for proteasome footprinting by mass-spectrometry analysis of proteasome-cleaved peptides (MAPP) ^[1]. MAPP offers, for the first time, direct analysis of the degradation products. Utilizing this system we have analyzed a clinical cohort of Non-Small Cell Lung Carcinoma (NSCLC) tumor samples and adjacent tissue controls to study the tumor degradome. Analysis by MAPP identified differences in the tumor tissue, some of which were not detected by standard proteomics of the same samples. Further, our analysis revealed altered metabolic pathways in the tumor tissue and offer novel targets for therapeutic intervention in NSCLC.

Methods:

The cohort included lung adenocarcinoma samples with matching adjacent tissue from nine patients, obtained from the Midgam biobank. MAPP was performed as previously described¹ by cellular crosslinking followed by immunopurification of cellular proteasomes with nascent cleaved peptides. Peptides were then eluted from proteasomes and analyzed by MS/MS. In parallel to MAPP, we also performed bottom-up tryptic proteomics from whole cell extract, thereby assessing the abundance of proteins in the same samples as well as their degradation profiles.

Results:

We found that there was a significant increase in the number of proteasome-generated peptides identified in the tumor samples as compared to the adjacent control. This correlates with an increase in proteasome abundance in the tumor. Intriguingly, there were many proteins which had a change in degradation level that was observed across the majority of the patients in the cohort. Key proteins in several pathways, some of which are already known to be altered in NSCLC such as glycolysis and glucose metabolism, were also misdegraded in the samples analyzed. Finally, there were many proteins which were not detected as degraded in the adjacent tissue but were highly degraded in a majority of tumor samples. Importantly, these differentially degraded proteins would have been missed by proteomics from whole cell extracts or RNAseq alone. These proteins are potential targets for cancer vaccines as they are present and degraded only in the tumor tissues.

Conclusion:

Taken together, MAPP offers a broadly applicable method to facilitate the study of cellular degradation in various human pathologies involving changes in proteasomal degradation, including cancer. The ability to use MAPP in clinical settings with small sample quantities promised to push the forefront of proteomics-based personalized medicine, highlighting new drug targets that are crucial to disease pathogenesis.

[1] Wolf-Levy, H., Javitt, A., Eisenberg-Lerner, A. et al. Revealing the Cellular Degradome by Mass Spectrometry Analysis of Proteasome-Cleaved Peptides. Nature Biotechnology. in Print (2018)