PMID: PMC6145689-2

 

    Legend: Gene, Sites

Title : In this study, 1,310 de-N-glycosylated peptides , 954 intact N-glycopeptides and 887 desialylated but otherwise intact O-glycopeptides belonging to a total of 788 glycoproteins were identified

Abstract :
  1. This deep coverage should be evaluated in the light of past efforts in this space including a study by Halim et al. [46], which showed the characterization of 58 N- and 63 O-glycopeptides from 53 urinary glycoproteins , Saraswat et al. [47] who reported 51 N-glycosylation sites belonging to 37 glycoproteins from the urinary exosomes and our own past study of urine i.e. Kawahara et al. [27] reporting 472 unique N-glycosylation sites covering 256 urinary glycoproteins
  2. Thus, this present study clearly represents the deepest coverage of the N- and O-glycoproteome of human urine to date
  3. One of the technical issues associated with urine analysis is the high inter- and intra-individual variability [6], which was previously shown to account for 48% and 66% of CV variation, respectively [48]
  4. In addition, the multi-step sample preparation and LC-MS/MS acquisition are likely sources of technical variation [49]
  5. We took advantage of the available TMT labeling technology, which not only allowed multiplexing of samples to reduce the LC-MS/MS acquisition time, but also minimized the technical variation from the sample handling [49], especially the peptide enrichment and pre-fractionation, while simultaneously facilitating more accurate quantification of glycopeptides and non-modified peptides [50]
  6. Due to the relative low number of biological replicates and high variance between the individuals, our study was limited to a non-stringent threshold of q < 0.25 for the peptide FDRs to determine differentially abundant glycoproteins in PCa and BPH urine
  7. However, by validating the levels of specific intact glycopeptides by PRM-MS/MS we significantly enhanced the confidence in these observations
  8. An important limitation of the chosen workflow is that, in complex mixtures, co-isolation of multiple precursor ions is the most acknowledged limitation of quantitative proteomics using isobaric labels
  9. This shortcoming reduces the precision and accuracy of the quantification [51, 52]
  10. It was demonstrated that due to this interference problem, the actual abundance ratios are typically underestimated [49]
  11. Using precursor intensity fraction (PIF) tool available in MaxQuant [53], we filtered peptides that clearly suffer from co-isolation of other peptide precursor ions
  12. Glycans facilitate and contribute to many different aspects of tumor progression, including proliferation, invasion, angiogenesis and metastasis [54]
  13. In cancer, protein glycosylation is dynamically regulated due to altered expression of glycan-modifying enzymes [55, 56]
  14. Wang et al. [57] showed high expression of α1,6-fucosyltransferase (FUT8 ) in tumor tissue from patients with metastatic and aggressive primary PCa and was positively correlated with PCa with high Gleason scores
  15. The N-glycosylation of proteins was demonstrated previously to be altered in urine [58], tissues [21] and serum [59] from PCa patients as well as in PCa cell lines [20]
  16. Interestingly, using meta-analysis of publicly available transcriptomic data from different PCa studies, Barfeld et al. [60] reported a concise 33 gene signature with biological enrichment for protein glycosylation, which discriminates between PCa and BPH across multiple transcript detection platforms and sample types
  17. Meany et al. [59] reported that direct analysis of PSA N-glycosylation in sera may be able to improve the sub-optimal specificity of PSA as a PCa marker
  18. Altered glycosylation of PSA isolated from PCa serum and/or seminal plasma relative to PSA glycosylation from controls was repeatedly observed by Tabares et al. [61], Llop et al. [62] and Ohyama et al. [63]
  19. Collectively, these studies clearly support the association of altered of protein glycosylation with PCa
  20. Although glycoproteomics studies have previously been used to explore PCa [20–22], the quantitative information relating to site-specific N- and O-glycosylation and the glycan com positions were not reported in those studies
  21. We demonstrated that differentially abundant glycopeptides discriminate more accurately between PCa and BPH groups than differentially abundant non-modified peptides
  22. Thus, we argue that a selected panel of glycopeptides , but not their corresponding non-glycosylated peptides from the same protein , may serve as candidate biomarkers for PCa detection
  23. We demonstrated that the malignant processes associated with PCa are more frequently reflected directly in the glycosylation of proteins as oppose to the aberrations in the expression level or the glycosylation site occupancy of those proteins
  24. Although we were limited to investigating a relative small patient cohort, the potential of intact glycopeptides to stratify patient groups can be explored in follow-up studies using larger cohort of patients once the glycoproteomics technology matures further allowing more streamlined data collection and interpretation of a larger patient cohort
  25. Among the PCa-associated glycopeptides , we targeted the N-glycosylation of prostaglandin-H2 D-isomerase ( PTGDS ) and the O-glycosylation of CD59 using PRM analysis
  26. Interestingly, Davalieva et al. [40] and Ahmad et al. [7] showed that these two glycoproteins are over-expressed in the urine of PCa patients
  27. Herein, we complement their observations by reporting that PTGDS and CD59 carry aberrant glycosylation at defined positions in PCa urine
  28. CD59 is a glycosyl-phosphatidylinositol ( GPI )-anchored cell membrane glycoprotein that inhibits complement-mediated cell lysis by preventing full assembly of the membrane attack complex (MAC) on host cells, and thus might confer immune resistance to tumor cells [42]
  29. High expression of CD59 protein was associated with higher Gleason scores and higher pT stages in PCa [41]
  30. It was demonstrated that prostasomes, which are secretory granules produced, stored, and released by the glandular epithelial cells of the prostate, had higher expression of CD59 than those of normal cells [64]
  31. CD59 glycosylation was previously characterized and several roles for the glycans, including spacing and orienting CD59 on the cell surface and protecting the molecule from proteases were proposed based on structural data [65]
  32. Prostaglandin-H2 D-isomerase was identified in human seminal plasma [66]
  33. Prostaglandin D2 (PGD2) and prostaglandin D2 synthase ( PTGDS ) is involved in the regulation of testis tissue differentiation [67, 68]
  34. PGD2, which is synthesized by PTGDS in many organs, has been implicated as a signaling molecule in the mediation or regulation of various biological processes, including tumorigenic process in PCa [69]
  35. It was shown that PTGDS activity is defined by post-translational modifications, and point mutation in a glycosylation site ( Asn51 ) inhibited L-PGDS-induced apoptosis and caspase 3 activity [70]
  36. Interestingly, we identified a significant lower abundance in the Asn51 glycoform HexNAc(4)Hex(5)Fuc(1)NeuAc(2) and HexNAc(5)Hex(6)Fuc(2) in PCa compared to BPH urine (Table 1)
  37. The quantitative analysis of intact glycoproteins opens novel avenues for assessing the dysregulation of glycoproteins in PCa and tie such information with aberrant glycoprotein function as drivers or passive by-products of tumour development and progression
  38. The quantification of specific peptides glycoforms is challenging due to the low ionization efficiency of glycan-modified peptides , as well as the lack of glycopeptides reference spectra
  39. Moreover, multiple glycoforms (micro-heterogeneity) are detected as separate glycopeptides , thus diminishing the signal intensity for each glycopeptide form
  40. Selected reaction monitoring ( SRM ) and parallel reaction monitoring (PRM) are currently the leading methods for targeted MS-based quantification of proteins
  41. Recently, SRM was applied to quantify candidate non-modified peptides biomarkers in expressed prostatic secretions (EPSs) from PCa and control patients [71]
  42. Song et al. [72] applied MRM to quantify intact glycopeptides in depleted human blood serum using the glycans oxonium ions as transitions
  43. However, to this date quantification of intact glycopeptides using targeted MS approaches is still poorly explored
  44. We showed PRM is a useful approach to quantify intact glycopeptides
  45. The number of MS/MS spectra recorder increased beyond a 20 fold by using PRM for targeted glycopeptide analysis, improving the accuracy of the identification and quantitation
  46. Integrating this approach with synthetic glycopeptides for normalization and absolute quantification would enhance value and utility of the PRM-MS/MS workflow even further
  47. We found the complement and coagulation cascades to be over-represented pathways in the set of urinary glycoproteins carrying differentially abundant site-specific glycoforms
  48. Interestingly, the same pathway was observed to be enriched in the meta-analysis from Barfeld et al. [60] which contained differentially expressed genes between localized PCa and benign prostate tissue, thus demonstrating that biological processes occurring in prostatic tissues may be reflected in urine
  49. Different coagulation disorders were reported during prostatic carcinoma evolution [73]
  50. The most frequent coagulation complication is the disseminated intravascular coagulation ( DIC ), which is a result of the release of pro-coagulant substances, such as tissue factors , into the bloodstream [74]
  51. This coagulation disorder was reported in many patients with PCa [75–77]
  52. We identified seven glycoproteins displaying aberrant site-specific glycosylation that are related to complement and coagulation cascade pathway
  53. The set of differentially abundant site-specific glycoforms belonging to these glycoproteins can be explored as candidate biomarkers for coagulation disorders that may be present during PCa progression
  54. Additionally, recent reports suggest that the complement elements can promote tumor growth in the context of chronic inflammation, immunosuppression, angiogenesis, and cancer cell signaling [78–80]
  55. Manning et al. also showed that human PSA , via its chymotrypsin-like serine protease activity, can modulate the complement system through degradation of iC3b to produce new C3 degradation fragments and through degradation of the complement protein C5 , thereby inactivating the complement cascade [81]
  56. In summary, this study reports several innovative approaches advancing our pursuit of reliable and sensitive urinary biomarker for PCa discovery and stratification from BPH by 1) providing the largest coverage of the urinary N- and O-glycoproteome to date, 2) providing a panel of 56 N-glycopeptides derived from aberrantly expressed glycoproteins in PCa urine representing an exciting collection of potential candidate biomarkers for discrimination of PCa from BHP and by 3) achieving confident structural characterization including the peptide carrier identity, site annotation, and glycan com position as well as quantitative validation of the panel of glycopeptide candidate markers by the application of innovative LC-MS/MS and PRM approaches of intact glycopetides
  57. This study opens new promising avenues for using urinary glycoproteins , a hitherto largely untapped resource, as candidate biomarkers for PCa detection
Output (sent_index, trigger, protein, sugar, site):
  • 0. N-glycopeptides, , -, -, N-glycopeptides
  • 0. O-glycopeptides, , -, -, O-glycopeptides
  • 0. de-N-glycosylated, , -, -, peptides
  • 0. desialylated, , -, -, O-glycopeptides
  • 0. glycoproteins, , glycoproteins, -, -
  • 1. N-glycosylation, , -, -, sites
  • 1. O-glycopeptides, , glycoproteins, -, N- and 63 O-glycopeptides
  • 1. glycoproteins, , glycoproteins, -, -
  • 15. N-glycosylation, , proteins, -, -
  • 18. glycosylation, , PSA, -, -
  • 21. glycopeptides, , -, -, glycopeptides
  • 22. glycopeptides, , -, -, glycopeptides
  • 22. non-glycosylated, , -, -, peptides
  • 23. glycosylation, , -, -, site
  • 23. glycosylation, , proteins, -, -
  • 23. occupancy, , proteins, -, site
  • 24. glycopeptides, , -, -, glycopeptides
  • 25. N-glycosylation, , CD59, -, -
  • 25. N-glycosylation, , PTGDS, -, -
  • 25. N-glycosylation, , prostaglandin-H2 D-isomerase, -, -
  • 25. O-glycosylation, , CD59, -, -
  • 25. O-glycosylation, , PTGDS, -, -
  • 25. O-glycosylation, , prostaglandin-H2 D-isomerase, -, -
  • 25. glycopeptides, , -, -, glycopeptides
  • 26. glycoproteins, , glycoproteins, -, -
  • 27. glycosylation, , -, -, positions
  • 28. glycoprotein, , CD59, -, -
  • 28. glycoprotein, , glycoprotein, -, -
  • 35. glycosylation, , -, -, Asn51
  • 35. glycosylation, , -, -, site
  • 36. Asn51, , -, the Asn51 glycoform HexNAc(4)Hex(5)Fuc(1)NeuAc(2), Asn51
  • 37. glycoprotein, , glycoprotein, -, -
  • 37. glycoproteins, , glycoproteins, -, -
  • 38. glycoforms, , -, -, peptides
  • 38. glycopeptides, , -, -, glycopeptides
  • 39. glycopeptide, , -, -, glycopeptide
  • 39. glycopeptides, , -, -, glycopeptides
  • 42. glycopeptides, , -, -, glycopeptides
  • 43. glycopeptides, , -, -, glycopeptides
  • 44. glycopeptides, , -, -, glycopeptides
  • 45. glycopeptide, , -, -, glycopeptide
  • 46. glycopeptides, , -, -, glycopeptides
  • 47. glycoproteins, , glycoproteins, -, -
  • 5. glycopeptides, , -, -, glycopeptides
  • 52. glycoproteins, , glycoproteins, -, -
  • 53. glycoproteins, , glycoproteins, -, -
  • 56. N-glycopeptides, , -, -, N-glycopeptides
  • 56. glycopeptide, , -, -, glycopeptide
  • 56. glycoproteins, , glycoproteins, -, -
  • 57. glycoproteins, , glycoproteins, -, -
  • 6. glycoproteins, , glycoproteins, -, -
  • 7. glycopeptides, , -, -, glycopeptides
Output(Part-Of) (sent_index, protein, site):
  • 1. glycoproteins, N- and 63 O-glycopeptides
  • 22. protein, peptides
*Output_Site_Fusion* (sent_index, protein, sugar, site):
  • 35. 788, -, Asn51
  • 36. 788, the Asn51 glycoform HexNAc(4)Hex(5)Fuc(1)NeuAc(2), Asn51

 

 

Protein NCBI ID SENTENCE INDEX