PMID: PMC6145689-1-3

 

    Legend: Gene, Sites

Title : HILIC HPLC pre-fractionation enhances the coverage of the urinary glycoproteome

Abstract :

1. In attempts to reach an even greater coverage of the urinary glycoproteome, off-line HILIC HPLC was implemented in the workflow to pre-fractionate the TiO2 SPE retained glycopeptide fraction
2. Endo- (PNGase F) and exo- (broad specificity sialidase ) glycosidase treatments were also introduced to render the HILIC peptide fractions more amendable to LC-MS/MS detection; it is commonly known that such de-N-glycosylated and desialo-O-glycopeptides are easier to identify than their native counterparts, albeit with less structural information obtained [33, 34]
3. Interesting, the de-N-glycosylated peptides eluted early in the HILIC HPLC gradient and were efficiently separated from the highly retained desialo-O-glycopeptides (Figure 2A)
4. When combined with Orbitrap Q-Exactive HF HCD-MS/MS detection, the HILIC HPLC pre-fractionation facilitated an unprecedented coverage of the urinary glycoproteome by the identification of a total of 1,217 N-glycosylation sites from 696 N-glycoproteins and the detection of 887 desialo-O-glycopeptides from 160 O-glycoproteins (Figure 2B and 2C and Supplementary Tables 7–8)
5. In total, 1,310 N-glycosylation sites were identified by combining the de-N-glycosylated peptides enriched by HILIC SPE and TiO2 SPE and pre-fractionated by HILIC HPLC (Supplementary Tables 2, 7)
6. We then compared our coverage of the urinary N-glycosylation sites in PCa and BPH urine to a SWATH based identification of the N-glycosylation sites reported in normal prostate, non-aggressive, aggressive and metastatic PCa tumor tissues [21]; a substantial overlap of 337 unique glycosylation sites (25%) and 321 N-gycoproteins (44%) was observed (Supplementary Figure 2A–2B)
7. This result indicates that almost half of the glycoproteins expressed in prostate tissues appear to be mirrored in the excreted urine
8. We then compared our urinary N-glycoproteome coverage to the N-glycoproteome of two PCa cell lines LNCap and PC3 reported by Shah et al. [20]
9. In total, 134 N-glycoproteins (11%) were found to be common between our two data sets (Supplementary Figure 2B)
10. Interestingly, the urinary glycoproteins identified in our study had a closer resemblance to the glycoproteome of PCa tissues than the glycoproteome of the PCa cell lines

Output (sent_index, trigger, protein, sugar, site):

• 1. glycopeptide, , -, -, glycopeptide
• 10. glycoproteins, , glycoproteins, -, -
• 2. desialo-O-glycopeptides, , -, -, desialo-O-glycopeptides
• 3. de-N-glycosylated, , -, -, peptides
• 3. desialo-O-glycopeptides, , -, -, desialo-O-glycopeptides
• 4. N-glycoproteins, , N-glycoproteins, -, -
• 4. N-glycosylation, , -, -, sites
• 4. O-glycoproteins, , O-glycoproteins, -, -
• 4. desialo-O-glycopeptides, , O-glycoproteins, -, desialo-O-glycopeptides
• 5. N-glycosylation, , -, -, sites
• 5. de-N-glycosylated, , -, -, peptides
• 6. N-glycosylation, , -, -, sites
• 6. glycosylation, , -, -, sites
• 6. reported, , -, -, sites
• 7. glycoproteins, , glycoproteins, -, -
• 9. N-glycoproteins, , N-glycoproteins, -, -

Output(Part-Of) (sent_index, protein, site):

• 4. N-glycoproteins, sites
• 4. O-glycoproteins, desialo-O-glycopeptides

*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

Protein	NCBI ID	SENTENCE INDEX