N- and O-Glycomics from Minor Amounts of Formalin-Fixed, Paraffin-Embedded Tissue Samples.

ABSTRACT

The availability of well-defined samples in sufficient numbers represents a major bottleneck for any biomarker related research. The utilization of preserved, archived and clinically well-described samples therefore holds a great potential to bridge this gap. This chapter describes a universal workflow for the comprehensive characterization of N- and O-glycans released from whole formalin-fixed, paraffin-embedded tissue sections, including an option for further partitioning using laser microdissection of specific tissue areas/cell populations. Glycoproteins are extracted and subsequently immobilized onto a PVDF membrane prior enzymatic release of N-glycans. Following N-glycan retrieval O-glycans are released using reductive ß-elimination from the same sample spot, significantly reducing the required amount of starting material. Released and reduced glycan structures are characterized using porous graphitized carbon liquid chromatography online coupled to an electrospray ionization-ion trap mass spectrometer. This technique provides information on the relative abundances of individual glycans along with detailed structural information, including isomer differentiation and functional epitope characterization of N- and O-glycans obtained from minimal amounts of tissue down to a few thousand cells.