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Site-specific characterization of N-linked glycosylation in human urinary glycoproteins and endogenous glycopeptides

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Abstract

Glycosylation is a very important post-translational modification involved in various cellular processes, such as cell adhesion, signal transduction and immune response. Urine is a rich source of glycoproteins and attractive biological fluid for biomarker discovery, owing to its availability, ease of collection, and correlation with pathophysiology of diseases. Although the urinary proteomics have been explored previously, the urinary glycoproteome characterization remains challenging requiring the development and optimization of analytical and bioinformatics methods for protein glycoprofiling. This study describes the high confident identification of 472 unique N-glycosylation sites covering 256 urinary glycoproteins. Besides, 202 unique N-glycosylation sites were identified in low molecular weight endogenous glycopeptides, which belong to 90 glycoproteins. Global site-specific characterization of the N-linked glycan heterogeneity was achieved by intact glycopeptide analysis, revealing 303 unique glycopeptides most of them displaying complex/hybrid glycans composed by sialic acid and fucose. These datasets consist in a valuable resource of glycoproteins and N-glycosylation sites found in healthy human urine that can be further explored in different disorders, in which the N-linked glycosylation may be aberrant.

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Acknowledgments

CNPq (GP 441878/2014-8) and FAPESP (GP: 2014/06863-3), Rebeca Kawahara is supported the Capes, PNPD, and FAPESP (2015/02866-0). Joyce Saad is supported by the “Programa Unificado de Bolsas de Estudo”. The facility Biomass at CEFAP-USP is acknowledged for the analyses.

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  1. GlycoProteomics Laboratory, Departamento de Parasitologia, ICB, University of São Paulo, São Paulo, Brazil

    Rebeca Kawahara, Joyce Saad, Claudia Blanes Angeli & Giuseppe Palmisano

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  1. Rebeca Kawahara
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  4. Giuseppe Palmisano
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Electronic Supplementary Material

Supplemental Figure 1

Tissue localization enrichment analysis in the list of identified urinary glycoproteins (PDF 36 kb)

Supplemental Figure 2

Manual verification of the Y1 + 16 Da in the MS/MS spectra from intact glycopeptides identified with NeuGc. The Y1 ion +16 Da is indicated by the red arrow. (PDF 248 kb)

Supplemental Figure 3

Charge state distribution of intact glycopeptides identified in each glycan composition class. The percentage was calculated based on the number of glycopeptides with charge +2, +3, +4 and >4 in each class to the total number of glycopeptides identified in each class. (PDF 15 kb)

Supplemental Figure 4

Overlap between the number of peptide sequences identified in the formerly N-linked peptides generated after PNGase F treatment and intact glycopeptides (with 0 % FDR) identified using Byonic software. (PDF 12 kb)

Supplemental Figure 5

Examples of annotated spectra for N-linked glycopeptides identified in urinary glycoproteins (PDF 62 kb)

Supplemental Figure 6

Sequence recognition motif in endogenous N-glycopeptides. N-glycosylation consensus sequence as derived using MotifX. A) MotifX analysis centered on the NxT glycosylation motif. B) MotifX analysis centered on the NxS glycosylation motif. C) Sequence2logo analysis obtained using the endogenous peptide sequences with six aminoacids length. (PDF 93 kb)

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Kawahara, R., Saad, J., Angeli, C.B. et al. Site-specific characterization of N-linked glycosylation in human urinary glycoproteins and endogenous glycopeptides. Glycoconj J 33, 937–951 (2016). https://doi.org/10.1007/s10719-016-9677-z

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