Formalin-fixed, paraffin-embedded (FFPE) tissues are banked in great numbers as a cost effective and simple way to preserve clinical specimens for study at a later time. The preservation method cross-links proteins and dehydrates cells, but primarily maintains the structural integrity of the tissue, making it a valuable resource for clinical assessment. Molecular analysis of FFPE tissue can be challenging due to potential chemical artifacts leftover from the preservation process, although recent developments in sample preparation and analytical techniques have helped to make FFPE tissue an asset for biomarker discovery. In particular, advancements in mass spectrometric techniques have allowed a more comprehensive characterization of a tissue"s proteome, including post-translational modifications (PTM). Glycosylation is one class of PTM that has been shown to be informative of certain disease outcomes. Global profiling of the glycoproteome can help to elucidate the complex signature specific to a biological state.

Here, we present a method to capture and analyze the glycoproteome from FFPE tissue samples that includes mechanical disruption and heat induced un-crosslinking of the tissue, detergent-based extraction and digestion of the proteome, and enrichment of glycopeptides through hydrophilic interaction chromatography. We estimate that ~ 10-12 mm³ of tissue, sliced into 10 μm sections, is sufficient material to produce a single replicate analysis of the glycoproteome. We compare, in three technical replicates, the glycoproteomic signature for a piece of FFPE tumor tissue from renal cell carcinoma (RCC) to a control of normal adjacent tissue (NAT) for the same individual subject and quantify over 2,000 unique protein groups and over 1,500 unique N-glycopeptides (derived from 169 glycoproteins) by LC-MS/MS through data dependent acquisition (DDA). We find more than 200 glycopeptides increased with statistical significance (p-value ≤ 0.05) in abundance in RCC compared to NAT, and more than 150 glycopeptides decreased with statistical significance (p-value ≤ 0.05) in abundance. Aggregate analysis of glycan subtypes reveal increased fucosylation in RCC compared to NAT. Altogether, we demonstrate a platform for glycoproteomic analysis of FFPE tissue with reproducible, quantitative results. This presents a unique opportunity to generate valuable insights into a number of diseases through the analysis of archived specimens.