PMID: PMC6145689-1-5

 

    Legend: Gene, Sites

Title : Comparative quantitative glycoproteomics to identify unique site-specific glycosylation signatures in PCa urine

Abstract :
  1. The datasets of de-N-glycosylated peptides and desialo-O-glycopeptides generated after TiO2 SPE-based glycopeptide enrichment and HILIC HPLC pre-fractionation as well as the intact N-glycopeptides identified after HILIC SPE and TIO2 SPE-based glycopeptide enrichments was used to quantitatively compare the glycosylation profile of PCa and BPH urine using TMT reporter ion intensities
  2. In addition, to assess for altered protein levels, the intensities from the non-modified peptides (TiO2 SPE flow-through fraction) were also compared between PCa and BPH
  3. The ion intensities of all glycopeptides or non-modified peptides were summed, log2 transformed, and median normalization was applied
  4. One BPH sample showed different outlier behavior when comparing all samples using PCA and was therefore removed
  5. To reveal statistical differences in the abundance of glycopeptides from PCa (n = 5) and BPH groups (n = 4), the Limma R package was used [36]
  6. The total number of differentially abundant intact N-glycopeptides and desialo-O-glycopeptides , de-N-glycosylated peptides and non-modified peptides , are shown in Figure 2D and Supplementary Table 9
  7. We then performed unsupervised PCA to assess if these glycopeptides and non-modified peptides were able to discriminate PCa from BPH
  8. Excitingly, all differentially abundant peptides , including the intact N- and O-glycopeptides , de-N-glycosylated peptides and non-modified peptides , were able to almost completely separate the PCa and BPH groups (Supplementary Figure 4A)
  9. Using only the non-modified peptides that were significantly changing in abundance, an overlap between PCa and BPH groups was observed
  10. Interestingly, only the differentially abundant intact N-glycopeptides and desialo-O-glycopeptides separated completely the PCa and BPH groups in the PCA
  11. As a complementary data representation style, we performed clustering analysis using Euclidean distance and a heat map to visualize the altered levels of intact N-glycopeptides and desialo-O-glycopeptides
  12. Two large clusters of glycopeptides were observed containing 120 over-represented glycopeptides and 106 under-represented glycopeptides in PCa compared to BPH (Supplementary Figure 4B)
  13. These clusters perfectly separated the PCa samples from the BPH samples
  14. These results showed that the urinary glycoproteome provided better discrimination between the PCa and BPH groups than the non-modified peptides
  15. Parallel reaction monitoring (PRM), a targeted proteomics strategy [37], was performed to: 1) confirm the levels of some intact glycopeptides in PCa urine; 2) increase the number of peptide-spectrum matches ( PSM ) for the individual intact glycopeptides to improve the identification confidence and 3) improve the quantification accuracy and ion statistics of intact glycopeptides with aberrant levels in the PCa urinary glycoproteome
  16. The intact N-glycopeptide SVVAPATDG GLNLTSTFLR displaying the triantennary and core-fucosylated sialoglycoform HexNAc(5)Hex(6)Fuc(1)NeuAc(3) from prostaglandin-H2 D-isomerase ( PTGDS ) was present in 29 PRM-MS/MS spectra within the retention time 31.2–32.9 min
  17. The intact O-glycopeptide DLCNFNEQLENGGTSLSEK carrying the glycoform HexNAc(3)Hex(1)Fuc(1) from CD59 glycoprotein CD59 glycoprotein ( CD59 ) was present in 24 PRM-MS/MS spectra within the retention time 22.8–24.9 min
  18. In comparison, each of these two glycopeptides were only identified from two acquired MS/MS spectra in a data-dependent acquisition (DDA) mode (Supplementary Tables 4 and 6)
  19. The TMT reporter ion intensities from the identified spectra of each glycopeptide were summed and normalized to the sum of all TMT reporter ion intensities from each TMT10plex channel during the entire LC-MS/MS run
  20. Statistical differences were assessed by conventional t-test analyses
  21. The increased levels of the HexNAc(5)Hex(6)Fuc(1)NeuAc(3) N-glycopeptide from PTGDS and decreased levels of the HexNAc(3)Hex(1)Fuc(1) O-glycopeptide from CD59 in PCa compared to BPH urine was confirmed (Supplementary Figure 5)
  22. Example of annotated PRM-MS/MS spectra of these two intact N- and O-glycopeptides are shown in Supplementary Figure 6
  23. Importantly, seven non-modified peptides from CD59 and eight non-modified peptides from PTGDS were identified
  24. Most of these non-modified peptides were unaltered in abundance when comparing between the PCa and BPH urine (Supplementary Table 10)
  25. This indicates that the expression levels of these two glycoproteins are likely not altered in PCa, but that the glycosylation of them instead is the molecular feature undergoing regulation
Output (sent_index, trigger, protein, sugar, site):
  • 1. N-glycopeptides, , -, -, N-glycopeptides
  • 1. N-glycopeptides, , -, -, peptides and desialo-O-glycopeptides
  • 1. de-N-glycosylated, , -, -, peptides and desialo-O-glycopeptides
  • 1. desialo-O-glycopeptides, , -, -, peptides and desialo-O-glycopeptides
  • 1. glycopeptide, , -, -, glycopeptide
  • 1. used, , -, -, N-glycopeptides
  • 10. N-glycopeptides, , -, -, N-glycopeptides and desialo-O-glycopeptides
  • 10. desialo-O-glycopeptides, , -, -, N-glycopeptides and desialo-O-glycopeptides
  • 11. N-glycopeptides, , -, -, N-glycopeptides and desialo-O-glycopeptides
  • 11. desialo-O-glycopeptides, , -, -, N-glycopeptides and desialo-O-glycopeptides
  • 12. glycopeptides, , -, -, glycopeptides
  • 15. glycopeptides, , -, -, glycopeptides
  • 16. D-isomerase, , prostaglandin-H2 D-isomerase, the triantennary and core-fucosylated sialoglycoform HexNAc(5)Hex(6)Fuc(1)NeuAc(3), -
  • 16. N-glycopeptide, , -, -, N-glycopeptide
  • 16. core-fucosylated, , -, the triantennary and core-fucosylated sialoglycoform HexNAc(5)Hex(6)Fuc(1)NeuAc(3), -
  • 17. O-glycopeptide, , -, -, O-glycopeptide
  • 17. glycoprotein, , CD59 glycoprotein, the glycoform HexNAc(3)Hex(1)Fuc(1) from CD59 glycoprotein, -
  • 18. glycopeptides, , -, -, glycopeptides
  • 19. glycopeptide, , -, -, glycopeptide
  • 21. N-glycopeptide, , -, -, N-glycopeptide
  • 21. O-glycopeptide, , -, -, O-glycopeptide
  • 22. O-glycopeptides, , -, -, O-glycopeptides
  • 25. glycoproteins, , glycoproteins, -, -
  • 3. glycopeptides, , -, -, glycopeptides
  • 5. glycopeptides, , -, -, glycopeptides
  • 6. N-glycopeptides, , -, -, N-glycopeptides and desialo-O-glycopeptides
  • 6. N-glycopeptides, , -, -, peptides
  • 6. de-N-glycosylated, , -, -, N-glycopeptides and desialo-O-glycopeptides
  • 6. de-N-glycosylated, , -, -, peptides
  • 6. desialo-O-glycopeptides, , -, -, N-glycopeptides and desialo-O-glycopeptides
  • 7. glycopeptides, , -, -, glycopeptides
  • 8. O-glycopeptides, , -, -, O-glycopeptides
  • 8. de-N-glycosylated, , -, -, O-glycopeptides
  • 8. de-N-glycosylated, , -, -, peptides
Output(Part-Of) (sent_index, protein, site):
  • 23. CD59, peptides
  • 23. PTGDS, peptides
*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

 

Protein NCBI ID SENTENCE INDEX