Universal Solid-Phase Reversible Sample-Prep for Concurrent Proteome and N‑Glycome Characterization

We
describe a novel solid-phase reversible sample-prep (SRS) platform
that enables rapid sample preparation for concurrent proteome and
N-glycome characterization for nearly all protein samples. SRS utilizes
a uniquely functionalized, silica-based bead that has strong affinity
toward proteins with minimal to no affinity for peptides and other
small molecules. By leveraging this inherent size difference between
proteins and peptides, SRS permits high-capacity binding of proteins,
rapid removal of small molecules (detergents, metabolites, salts,
peptides, etc.), extensive manipulation including enzymatic and chemical
treatments on bead-bound proteins, and easy recovery of N-glycans
and peptides. SRS was evaluated in a wide range of samples including
glycoproteins, cell lysate, murine tissues, and human urine. SRS was
also coupled to a quantitative strategy to investigate the differences
between DU145 prostate cancer cells and its DIAPH3-silenced counterpart.
Previous studies suggested that DIAPH3 silencing in DU145 induced
transition to an amoeboid phenotype that correlated with tumor progression
and metastasis. In this pilot study we identified distinct proteomic
and N-glycomic alterations between them. A metastasis-associated tyrosine
kinase receptor ephrin-type-A receptor (EPHA2) was highly up-regulated
in DIAPH3-silenced cells, indicating a possible connection between
EPHA2 and DIAPH3. Moreover, distinct alterations in the N-glycome
were identified, suggesting cross-links between DIAPH3 and glycosyltransferase
networks.