PMID: PMC6145689-1-1

 

    Legend: Gene, Sites

Title : Quantitative glycoproteomics enable exploration of the urinary N- and O-glycoproteome

Abstract :
  1. Urine samples (∼2 ml) from six PCa and six BPH patients were obtained and investigated in this study
  2. The PCa group included only patients with clinically validated adenocarcinoma with Gleason score 7 (GS 7), which denotes intermediate grade PCa relative to GS 6 representing low-risk PCa suitable for an active surveillance program, and the more advanced GS 8–10 representing high-risk PCa cases that generally are referred to immediate treatment
  3. The serum PSA levels of the PCa patient group were 7.17 ± 3.02 ng/ml (n = 6) and 8.02 ± 7.30 ng/ml in the BPH patient group (n = 6)
  4. As expected, no significant difference was observed in the serum PSA levels between these two patient groups (p ≥ 0.05, n = 6, unpaired two-tailed t-test, Figure 1A) highlighting the shortcoming of this biomarker to accurately stratify PCa and BHP patients
  5. Lower yields of total urinary protein were observed from two biological replicates from both the PCa and BPH groups; hence, these protein-poor samples were combined (1:1, w/w) forming five biological replicates (n = 5) from each condition for comparative glycoproteomics
  6. Given the reasonably large protein starting material (∼100 µg/replicate), we applied a multi-faceted quantitative LC-MS/MS-based glycoproteomics strategy to obtain a deep coverage of the urinary glycoproteome and investigate for differentially abundant glycoproteins in PCa and BPH urine
  7. In short, after protein extraction, concentration and tryptic digestion of the 10 samples, the resulting peptide mixtures were labeled with an isobaric amine-reactive TMT10plex – peptides from the five PCa samples were labeled with TMT 126, 127N, 127C, 128N and 128C and peptides from the five BPH samples were labeled with TMT 129N, 129C, 130N, 130C and 131
  8. All TMT-labelled peptide samples were then mixed in 1:1 (w/w) relationships prior to a multi-faceted glycopeptide enrichment and pre-fractionation sample processing (Figure 1B)
  9. The glycopeptide enrichment was performed using two complementary solid phase extraction (SPE) strategies i.e. titanium dioxide (TiO2) SPE and HILIC SPE enrichment, to reach the deepest possible coverage of the urinary glycoproteome
  10. The sialic acid-retaining TiO2 SPE [28, 29] was included since we previously showed that urinary N-glycoproteins are highly sialylated [27]
  11. HILIC SPE is widely recognized to capture all glycopeptides displaying a minimum degree of hydrophilicity regardless of the nature of their conjugated glycan structure [30, 31]; however, this enrichment method may come short of quantitatively capturing O- and N-glycopeptides of very low hydrophilicity (unpublished observation)
  12. The TiO2 and HILIC SPE enriched glycopeptides were 1) analyzed directly in their intact form by high resolution LC-MS/MS on a Orbitrap Q-Exactive HF with HCD fragmentation , which provided information of the peptide carrier and site-specific glycan heterogeneity, or 2) treated simultaneously with N-glycosidase F ( PNGase F) and sialidase A to generate de-N-glycosylated and desialo-O-glycopeptides that are less challenging to characterize by LC-MS/MS
  13. Combining the latter approach with a similar Orbitrap Q-Exactive HF HCD based acquisition, provided N-glycosylation site information and site-specific glycan information of the O-glycosylation
  14. In addition, non-modified peptides that did not bind to the TiO2 SPE column (“flow through”) and PNGase F/sialidase-treated glycopeptides were pre-fractionated using off-line HILIC HPLC and the resulting fractions were analyzed by Orbitrap Q-Exactive HF HCD-MS/MS (Figure 1B)
Output (sent_index, trigger, protein, sugar, site):
  • 10. N-glycoproteins, , N-glycoproteins, -, -
  • 10. sialylated, , N-glycoproteins, -, -
  • 11. N-glycopeptides, , -, -, N-glycopeptides
  • 11. glycopeptides, , -, -, glycopeptides
  • 12. desialo-O-glycopeptides, , -, -, desialo-O-glycopeptides
  • 12. glycopeptides, , -, -, glycopeptides
  • 13. N-glycosylation, , -, -, site
  • 14. glycopeptides, , -, -, glycopeptides
  • 6. glycoproteins, , glycoproteins, -, -
  • 8. glycopeptide, , -, -, glycopeptide
  • 9. glycopeptide, , -, -, glycopeptide
Output(Part-Of) (sent_index, protein, site):
  • 14. PNGase, glycopeptides
*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

 

Protein NCBI ID SENTENCE INDEX