PMID: PMC6145689-1-2

 

    Legend: Gene, Sites

Title : Complementary TiO2 and HILIC SPE-based glycopeptide enrichment provide deep coverage of the urinary glycoproteome

Abstract :

1. Even without peptide pre-fractionation, the combined use of TiO2 and HILIC SPE facilitated the identification of 630 unique N-linked glycosylation sites belonging to 361 N-glycoproteins across the urine samples (Supplementary Figure 1A, and Supplementary Tables 1–2)
2. Partial overlap between the TiO2 and HILIC SPE enrichment approaches were observed when measured by the proportion of unique N-glycosylation sites (48%, 304/630) and the corresponding glycoproteins (58%, 211/361) detected in both sample preparation techniques (Supplementary Figure 1B–1C)
3. However, when assessed by the identified intact glycopeptides , the enrichment strategies displayed high complementarity: From a combined total of 954 N- and 210 O-glycopeptides identified with high peptide confidence (0% peptide FDR Pep-2D) [32] (Supplementary Figure 1D and Supplementary Tables 3–6), the degree of unique N- and O-glycopeptides identified from both the TiO2 and HILIC SPE preparations was 23% (216/954) and 10% (20/210), respectively (Supplementary Figure 1E–1F)
4. Due to the unbiased enrichment of the hydrophilic glycopeptides , HILIC SPE showed, as expected, a higher capture efficiency of N-glycopeptides compared to TiO2 SPE
5. In contrast, TiO2 retained a greater number of O-glycopeptides compared to HILIC SPE indicating a high degree of sialylation of the O-glycopeptides and an inability of HILIC SPE to retain such lowly hydrophilic glycopeptides
6. We also investigated the glycosylation features of the TiO2 and HILIC SPE enriched intact N-glycopeptides (Supplementary Figure 1G, Supplementary Tables 3–4) and O-glycopeptides (Supplementary Figure 1H, Supplementary Tables 5–6)
7. In agreement with our previous study [27], the urinary N-glycoproteins were predominantly carrying complex and hybrid type N-glycans with a high level of fucosylation and/or sialylation accounting for ∼85% of all identified intact N-glycopeptides (Supplementary Figure 1G)
8. Sialylated O-glycopeptides were also abundant (∼70%) and were, as expected, efficiently enriched using TiO2 SPE (Supplementary Figure 1H)
9. Collectively, these data clearly demonstrated that the parallel usage of HILIC and TiO2 SPE is highly beneficial to increase the coverage of the urinary N- and O-glycoproteome

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

• 0. glycopeptide, , -, -, glycopeptide
• 1. N-glycoproteins, , N-glycoproteins, -, -
• 1. glycosylation, , -, -, sites
• 2. N-glycosylation, , -, -, sites
• 2. glycoproteins, , glycoproteins, -, -
• 3. O-glycopeptides, , -, -, N- and 210 O-glycopeptides
• 3. O-glycopeptides, , -, -, O-glycopeptides
• 3. glycopeptides, , -, -, glycopeptides
• 4. N-glycopeptides, , -, -, N-glycopeptides
• 4. glycopeptides, , -, -, glycopeptides
• 5. O-glycopeptides, , -, -, O-glycopeptides
• 5. glycopeptides, , -, -, glycopeptides
• 5. sialylation, , -, -, O-glycopeptides
• 6. N-glycopeptides, , -, -, N-glycopeptides
• 6. O-glycopeptides, , -, -, O-glycopeptides
• 6. TiO2, , -, -, N-glycopeptides
• 7. N-glycopeptides, , -, -, N-glycopeptides
• 7. N-glycoproteins, , N-glycoproteins, -, -
• 8. O-glycopeptides, , -, -, O-glycopeptides

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

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

 

Protein	NCBI ID	SENTENCE INDEX