A Fully Integrated Spintip-Based Approach for Sensitive and Quantitative Profiling of Region-Resolved in Vivo Brain Glycoproteome

Region-
and cell type-resolved global proteome and specific post-translational
modifications (PTMs) profiling of tissues has drawn great attention
recently for interpreting the heterogeneous multicellular microenvironment
of various in vivo systems. Due to access to low microgram of proteins
and low abundance of glycoproteins, spatially resolved glycoproteome
analysis of in vivo tissue sections remains challenging. Several glycoproteomics
sample preparation strategies were established for processing microgram-level
of protein samples, but these strategies were not either fully integrated
or directly compatible with tissue samples when considering protein
extraction in strong lysis buffers. Moreover, these approaches mainly
focused on identification of glycosylation sites, but pay less attention
to quantification, all of which limit their applications. Here we
designed a fully integrated spintip-based glycoproteomic approach
(FISGlyco) which achieves all the steps of glycoprotein enrichment,
digestion, deglycosylation, and desalting in single spintip device.
Sample loss is significantly reduced, and the total processing time
is reduced to 4 h, while detection sensitivity and label-free quantification
precision is greatly improved. 607 N-glycosylation sites were successfully
identified and quantified from only 5 μg of mouse brain proteins.
By seamlessly combining with laser capture microdissection (LCM),
the first region-resolved N-glycoproteome profiling of four mouse
brain regions, including isocortex, hippocampus, thalamus, and hypothalamus,
was achieved, with 1875, 1794, 1801, and 1417 N-glycosites identified,
respectively. Our approach could be a generic approach for region
and even cell type specific glycoproteome analysis of in vivo tissue
sections.