Differential Glycosite Profiling-A Versatile Method to Compare Membrane Glycoproteomes.

Glycosylation is the most prevalent and varied form of post-translational protein modifications. Protein glycosylation regulates multiple cellular functions, including protein folding, cell adhesion, molecular trafficking and clearance, receptor activation, signal transduction, and endocytosis. In particular, membrane proteins are frequently highly glycosylated, which is both linked to physiological processes and of high relevance in various disease mechanisms. The cellular glycome is increasingly considered to be a therapeutic target. Here we describe a new strategy to compare membrane glycoproteomes, thereby identifying proteins with altered glycan structures and the respective glycosites. The workflow started with an optimized procedure for the digestion of membrane proteins followed by the lectin-based isolation of glycopeptides. Since alterations in the glycan part of a glycopeptide cause mass alterations, analytical size exclusion chromatography was applied to detect these mass shifts. N-glycosidase treatment combined with nanoUPLC-coupled mass spectrometry identified the altered glycoproteins and respective glycosites. The methodology was established using the colon cancer cell line CX1, which was treated with 2-deoxy-glucose-a modulator of N-glycosylation. The described methodology is not restricted to cell culture, as it can also be adapted to tissue samples or body fluids. Altogether, it is a useful module in various experimental settings that target glycan functions.

The shared frameshift mutation landscape of microsatellite-unstable cancers suggests immunoediting during tumor evolution.

The immune system can recognize and attack cancer cells, especially those with a high load of mutation-induced neoantigens. Such neoantigens are abundant in DNA mismatch repair (MMR)-deficient, microsatellite-unstable (MSI) cancers. MMR deficiency leads to insertion/deletion (indel) mutations at coding microsatellites (cMS) and to neoantigen-inducing translational frameshifts. Here, we develop a tool to quantify frameshift mutations in MSI colorectal and endometrial cancer. Our results show that frameshift mutation frequency is negatively correlated to the predicted immunogenicity of the resulting peptides, suggesting counterselection of cell clones with highly immunogenic frameshift peptides. This correlation is absent in tumors with Beta-2-microglobulin mutations, and HLA-A*02:01 status is related to cMS mutation patterns. Importantly, certain outlier mutations are common in MSI cancers despite being related to frameshift peptides with functionally confirmed immunogenicity, suggesting a possible driver role during MSI tumor evolution. Neoantigens resulting from shared mutations represent promising vaccine candidates for prevention of MSI cancers.