Figure 4.

Glycoproteogenomics, building on RNAseq-customized databases and glycomics for protein annotation, enables CD44 proteoforms identification in bladder cancer cell lines and tumors. A) Glycomics characterization of 5637 and T24 cells showed fucosylated and sialylated T antigens as main glycospecies. MALDI-MS analysis of permethylated benzyl-GalNAc glycans revealed [M+Na]⁺ main ions for fucosylated and sialylated T antigens in both cell lines. However, 5637 predominantly expressed mono-sialylated T antigens whereas di-sialylated structures were more abundant in T24 cells. Moreover, 5637 cells presented higher abundance and diversity of glycoforms extended beyond core 2 with different degrees of sialylation and fucosylation. B) SDS-PAGE gels and western blots for CD44 IPs highlighting the nature of the isoforms identified by nano-LC-M/MS in glycoproteogenomics settings. Briefly, CD44 immunoprecipitated from membrane extracts was separated by gradient SDS-PAGE and bands were excised and analyzed by nanoLC-HCD/CID-MS/MS using RNAseq-customized databases and glycomics data for isoforms annotation. The nature of the isoforms translated by reporter ions for constitute-variable junctions identified in each band and cell line was highlighted. CD44 heterogeneity was evident for the two cell lines. CD44s was found to be the main isoform in T24 cells. C) nano-LC-MS and HCD-MS/MS spectra for reporter ions v2-v3, e5-v3, e5-e15 (CD44s/st), and e2(partial)-e3(partial) CD44sol in T24 cells. For reporter ions we show the MS isotopic envelope, the corresponding HCD product ion spectra highlighting GalNAc oxonium ions, GalNAc cross-ring fragments, other glycan fragments, and y- and b-series peptide backbone ions that support protein annotation. The predicted glycopeptide sequence, including the nature of the glycans and glycosites annotation (whenever possible) was also presented. D) Glycosites identified for v2-v3, e5-v3 and e5-e15 reporter glycopeptides and corresponding glycans for 5637 and T24 cells. A higher number of glycosites were identified at the e5-e15 junction for T24 in comparison to 5637 cells. On the other hand, more glycosites were identified at the v2-v3 and e5-v3 junctions in 5637 cells. E) nano-LC-MS and HCD-MS/MS spectra for CD44 immunoprecipitated from CD44s^high tumors and areas of CD44-STn co-expression. SDS-PAGE and western blots showed a pattern similar to T24 cells, characterized by bands bellow 75 kDa. An HCD product ion spectrum for an CD44s-Tn specific glycopeptide sequence is presented. Identified and possible glycosites are identified in grey. The symbol * corresponds to amino acid modifications: C - carbamidomethyl and T - 2-amino-3-ketobutyric acid.