Lung adenocarcinoma (LUAC) accounts for approximately 40% of all lung cancer cases. Several driver mutation(s) contribute to the genetic heterogeneity of LUAC. LUAC that is wild type for the EGFR, KRAS, and ALK genes is commonly referred to as "triple wild-type" (triple wt). Triple wt lung cancer has a poor prognosis, so the molecular characterization of this cancer type is especially important. Increasing evidence suggests that aberrant protein glycosylation plays a crucial role in the pathogenesis and progression of lung cancer. Alterations in glycosylation patterns have been previously observed between healthy and cancerous samples as well as between different lung cancer subtypes. Therefore, our ultimate goal is to compare the glycosaminoglycan and N-glycoproteomic profiles of triple wt LUAC samples and those harbouring different mutations (EGFR, KRAS and ALK), approximately 100 samples altogether.

In the present pilot study our aim was the detailed proteomic and N-glycoproteomic characterization of the tumor and tumor adjacent regions of formalin-fixed paraffin embedded human lung tissue samples from a cohort of triple wt lung cancer patients (n=12). Following dewaxing and antigen retrieval of the tissue sections, on-surface tryptic digestion was performed on regions of interest, then glycopeptide enrichment was carried out using acetone precipitation. The pellet and supernatant fractions were analyzed separately, using different nanoHPLC-MS methods. Altogether 3376 proteins were quantified using MaxQuant, and 1626 unique glycopeptides were identified corresponding to 865 glycosylation sites on 365 glycoproteins using GlycReSoft.

The protein expression profiles of the triple wt tumor and adjacent normal regions were found to be markedly different. Significant differences were identified in the expression levels of 1066 proteins, and several biological processes were found to be enriched (for both up- and downregulated genes) based on Fast Gene Set Enrichment Analysis (FGSEA). We have identified 28 glycopeptides with differences in abundance between tumor and adjacent normal tissue. Several of the corresponding glycoproteins showed significant differences in protein expression levels suggesting that changes detected in glycopeptide abundance can be attributed to a combination of changes in both glycoprotein expression, and glycosylation. To the best of our knowledge this the first study to analyze the N-glycosylation of glycoproteins of triple wt lung cancer and could be a valuable resource for further research regarding the potential of diagnostic glycoprotein markers, and drug targets as well.

Support of the National Research, Development and Innovation Office (OTKA FK 131603, and OTKA K-147226) and the Lendület (Momentum) Program of the Hungarian Academy of Sciences is acknowledged.