Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Glycoproteomic analysis of human colorectal adenoma tissue (#751)

Krishnatej Nishtala 1 , Howard CH Yim 2 , Emad El-Omar 2 , Nicolle H Packer 1
  1. Dept of Molecular Sciences, Macquarie University, Sydney, NSW, Australia
  2. St George & Sutherland Clinical School, UNSW, Sydney, NSW, Australia

Colorectal cancer develops over a few decades with genetic mutations occurring in the mucosal cells leading to their autonomous proliferation into premalignant adenomatous polyps which further become cancerous. Hence our goal was to characterise the disease associated changes in the glycoproteome of colorectal adenoma tissue using mucin-depleted human colon polyp proteins.

Tissue lysates from biopsies of adenomatous polyps and matched adjacent non-adenomatous tissue from the colon of patients diagnosed with dysplasia were precipitated by the addition of guanidine hydrochloride (6M GuHCl), which is the reported method of choice for MUC 2 enrichment (Larsson et al., Glycobiol., 2009). The pellet and the supernatant were analysed for their proteome. An equal fraction of the tissue was processed without GuCl precipitation to capture the whole proteome. Affinity enrichment by ZIC-HILIC was carried out on tryptic digests of the total tissue and the GuHCl supernatant for capturing the non-mucin glycopeptides. The samples were analysed on a Thermo QExactive HFX Orbitrap mass spectrometer. The data were analysed using Proteome Discoverer 2.2 and Byonic v3.3 for glycopeptide identification.

Proteomic analysis of the 6M GuHCl precipitated proteins from the tissue lysates identified MUC2 (104 unique peptides) as the expected predominant colonic mucin but MUC5AC, MUC5B, Microfibril associated glycoprotein 4, Transmembrane glycoprotein NMB and Vitronectin were also abundant. These proteins were either not identified or of very low abundance in the whole tissue proteome. Cytosolic proteins comprised the majority of the GuCl supernatant fraction indicating efficient total mucin enrichment by GuCl precipitation. Byonic analysis of the ZIC-HILIC enriched fraction of the trypsin-digested GuHCl supernatants identified around 550 glycopeptides (O-glycosylated: 145, N-glycosylated: 320 and N & O glycosylated: 85) corresponding to about 200 proteins in the colorectal polyp. This approach was used to characterise the non-mucin related glycoproteome of colorectal adenoma dysplasia compared to matched normal tissue.