Workflow for Combined Proteomics and Glycomics Profiling from Histological Tissues

Extracellular matrixes comprise glycoproteins, glycosaminoglycans and proteoglycans that order the environment through which cells receive signals and communicate. Proteomic and glycomic molecular signatures from tissue surfaces can add diagnostic power to the immunohistochemistry workflows. Acquired in a spatially resolved manner, such proteomic and glycomic information can help characterize disease processes and be easily applied in a clinical setting. Our aim toward obtaining integrated omics datasets was to develop the first workflow applicable for simultaneous analysis of glycosaminoglycans, N-glycans and proteins/peptides from tissue surface areas as small as 1.5 mm in diameter. Targeting small areas is especially important in the case of glycans, as their distribution can be very heterogeneous between different tissue regions. We first established reliable and reproducible digestion protocols for the individual compound classes by applying standards on the tissue using microwave irradiation to achieve reduced digestion times. Next, we developed a multienzyme workflow suitable for analysis of the different compound classes. Applicability of the workflow was demonstrated on serial mouse brain and liver sections, both fresh frozen and formalin-fixed. The glycomics data from the 1.5 mm diameter tissue surface area was consistent with data published on bulk mouse liver and brain tissues, which demonstrates the power of the workflow in obtaining combined molecular signatures from very small tissue regions.