Protein glycosylation is a ubiquitous post-translation modification well known to functionally influence many biological processes such as immune modulation, cell-cell interactions or signal transduction. The composition of a cell's glycome is dependant of numerous factors such as age, health or disease state, and several examples successfully demonstrated the applicability of individual glycosylation alterations as specific disease markers. This makes glycosylation a prime target for biomarker discovery, nevertheless the availability of well-defined clinical specimens in sufficient numbers often represents a serious obstacle in glyco-biomarker analyses. The possibility to use formalin-fixed paraffin-embedded (FFPE) clinical specimens presents an attractive alternative for glyco-biomarker research given that any glyco-epitopes remain unaltered and sufficient glycan amounts can be obtained from conventional FFPE tissue sections frequently used by pathologists.

Here we present an approach for the in-depth glycomic profiling of both, N-and O-glycans starting from single FFPE tissue sections as thin as 3 µm. Porous graphitized carbon (PGC) nanoLC ESI-MS/MS provides excellent isomer separation, semi-automated structure identification via spectral matching and relative quantitation of individual structures. Unstained as well as hematoxylin and eosin (HE) stained FFPE tissue samples derived from routinely taken clinical preparations were analysed and compared to their corresponding frozen counterparts to identify any potential FFPE and staining induced alterations on the N- and O-glycome. Furthermore, N- and O-glycans could successfully be retrieved from cells isolated by laser micro dissection from FFPE sections (ranging from 10000 down to 1000 cells). Overall around 90 individual glycan structures including structural isomers were identified from human hepatocellular carcinoma cells and non-tumour liver tissue. Major glyco-epitopes containing neuraminic acid and/or fucose were not affected in their structural integrity. These results emphasise the potential provided by FFPE tissue slides as a valuable source for glyco-biomarker studies from clinical specimens.