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StrucGP: de novo structural sequencing of site-specific N-glycan on glycoproteins using a modularization strategy

Nature Methods volume 18, pages 921–929 (2021)Cite this article

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Abstract

Precision mapping of glycans at structural and site-specific level is still one of the most challenging tasks in the glycobiology field. Here, we describe a modularization strategy for de novo interpretation of N-glycan structures on intact glycopeptides using tandem mass spectrometry. An algorithm named StrucGP is also developed to automate the interpretation process for large-scale analysis. By dividing an N-glycan into three modules and identifying each module using distinct patterns of Y ions or a combination of distinguishable B/Y ions, the method enables determination of detailed glycan structures on thousands of glycosites in mouse brain, which comprise four types of core structure and 17 branch structures with three glycan subtypes. Owing to the database-independent glycan mapping strategy, StrucGP also facilitates the identification of rare/new glycan structures. The approach will be greatly beneficial for in-depth structural and functional study of glycoproteins in the biomedical research.

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Fig. 1: Principle of the new method for structural interpretation of site-specific N-glycans.
Fig. 2: The performance of StrucGP on structural analysis of site-specific N-glycans from standard glycoproteins.
Fig. 3: Site-specific N-glycan structure analysis of mouse brain.
Fig. 4: StrucGP reveals glycan isoforms and new/rare glycans in mouse brain.
Fig. 5: Error rate control and probability estimation of StrucGP results.
Fig. 6: Comparison of StrucGP with GPQuest v.2.0, Byonic v.3.0 and pGlyco v.2.0.

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Data availability

The mass spectrometry data, as well as all spectra for identified glycopeptides from different samples, have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository43 with the dataset identifier PXD025859.

Code availability

StrucGP was developed in the python language and the standalone software package can be downloaded at the Zenodo repository (https://doi.org/10.5281/zenodo.4925441)44.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China, grant nos. 91853123 (S.S.), 81773180 (S.S.), 21705127 (S.S.) and 81800655 (L.D.); National Key R&D Program of China, grant no. 2019YFA0905200 (S.S.) and China Postdoctoral Science Foundation, grant nos. 2019M653715 (L.D.), 2019TQ0260 (J.L.) and 2019M663798 (J.L.).

Author information

Author notes

  1. These authors contributed equally: Jiechen Shen, Li Jia, Liuyi Dang, Yuanjie Su.

Authors and Affiliations

  1. College of Life Science, Northwest University, Xi’an, China

    Jiechen Shen, Li Jia, Liuyi Dang, Yintai Xu, Bojing Zhu, Zexuan Chen, Jingyu Wu, Rongxia Lan, Zhifang Hao, Chen Ma, Ting Zhao, Ni Gao, Jieyun Bai, Yuan Zhi, Jun Li & Shisheng Sun

  2. School of Computer Science and Technology, Xidian University, Xi’an, China

    Yuanjie Su, Jie Zhang & Junying Zhang

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Contributions

S.S. and J.S. planned and designed the project. J.S., Y.S. and Jie Zhang developed the algorithm. L.J., R.L., Z.H., T.Z. and B.J. optimized protocols and prepared samples. B.Z., L.J., Z.H. and C.M. performed mass spectrometry analyses with MS parameter optimizations. J.S., S.S., J.L., Y.X., L.D., Z.C., J.W., C.M., N.G., J.B. and Y.Z. contributed to the data analyses and figure generation. Junying Zhang and S.S. coordinated the study. L.D., S.S. and J.S. prepared the manuscript with contributions from all coauthors.

Corresponding author

Correspondence to Shisheng Sun.

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The authors declare no competing interests.

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Peer review information Arunima Singh was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Workflow of StrucGP.

Workflow of StrucGP for glycan structure interpretation on intact glycopeptides. Related to Fig. 1a.

Extended Data Fig. 2 Glycan structures identified from standard glycoproteins.

The standard glycoproteins include (a) bovine RNase B, (b) bovine fetuin, (c) ovalbumin from chicken, and (d) human IgG including IgG1-4. e, An example of intact glycopeptide identification from fetuin using StrucGP. Related to Fig. 2b–e and Supplementary Table 1.

Extended Data Fig. 3 Site-specific N-glycan structure analysis of mouse brain.

a, Recognition and identification of four types of core structures in the mouse brain using their feature Y ion patterns. b, Determination of glycan subtypes in mouse brain using three feature Y ion patterns. Related to Fig. 3b,c.

Extended Data Fig. 4 Determination of branch glycan structures using feature B ions.

The branch glycan structures were identified in mouse brain and standard glycoproteins. The example spectra of each branch structure are shown in Supplementary Data 1.

Extended Data Fig. 5 Glycan structures identified in mouse brain.

A total of 600 N-glycan structures were identified in mouse brain. It is worth to mention that the location of each branch structure (such as α-2,3 or α-2,6 mannose) can’t actually be identified by StrucGP. Related to Fig. 3 and Supplementary Table 2.

Extended Data Fig. 6 Glycan isoforms sharing the same glycan compositions.

This figure shows glycan compositions that have identified at least seven glycan isoforms in the mouse brain. Related to Fig. 4a,b.

Extended Data Fig. 7 Spectra of glycopeptides with three glycan isoforms of N4H5F1 on the same peptide.

The peptide TLN#CSGAHVK was modified by three different isoforms of the glycan HexNAc4Hex5Fuc1 (N4H5F1). Upper: peptide sequences identification using a MS/MS spectrum of high HCD energy (HCD = 30%). Lower: glycan structure determination using MS/MS spectra of low HCD energy (HCD = 20%). Related to Fig. 4c.

Extended Data Fig. 8 Spectra of glycopeptides with two glycan isoforms of N4H5F1 on the same peptide.

The peptide GN#GTLITFHSAFQCCGK was modified by two different isoforms of the glycan HexNAc4Hex5Fuc1 (N4H5F1). Upper: peptide sequences identification using a MS/MS spectrum of high HCD energy (HCD = 33%). Lower: glycan structure determination using MS/MS spectra of low HCD energy (HCD = 20%). Related to Fig. 4c.

Extended Data Fig. 9 Glycan structures identified from five mouse tissues.

A total of 719 N-glycan structures were identified from five mouse tissues. The 25 mass spectrometry raw files (five raw files per mouse tissue) reported in the pGlyco 2.0 study were downloaded from the ProteomeXchange database.

Extended Data Fig. 10 Expression patterns of site-specific glycan structures in different mouse tissues.

The 25 mass spectrometry raw files reported in the pGlyco 2.0 study were downloaded from the ProteomeXchange database. a, Overall processes of intact glycopeptide analysis of five different mouse tissues. b, Percentages of identified spectra that had at least 99% probabilities of related substructures from five mouse tissue data. Comparison of glycan core structures (c) and glycan subtypes (d) among intact glycopeptides identified by StrucGP from five mouse tissues. *indicates mouse brain glycopeptides identified from this study. e, Comparison of branch structures among five mouse tissues. The percentages in this figure were calculated based on the numbers of unique glycopeptides. Different isomers can be distinguished by specific feature B ions, the same method as shown in Extended Data Fig. 4.

Supplementary information

Supplementary Information

Supplementary Notes 1–5, Figs. 1–5 and Table 6.

Reporting Summary

Supplementary Tables

Supplementary Table 1. List of intact glycopeptides with detailed glycan structures identified from standard glycoproteins with and without exoglycosidase treatments using StrucGP. Supplementary Table 2. List of intact glycopeptides with detailed glycan structures identified from mouse brain using StrucGP. Supplementary Table 3. List of intact glycopeptides identified in exoglycosidase-treated mouse brain samples using StrucGP. Supplementary Table 4. List of intact glycopeptides with detailed glycan structures identified from Drosophila Schneider-2 cells using StrucGP. Supplementary Table 5. List of intact glycopeptides with detailed glycan structures identified from five mouse tissues by analyzing previously reported mass spectrometry data with StrucGP.

Supplementary Data

Determination of branch glycan structures in mouse brain and standard glycoproteins using feature B ions with further validation using Y ions.

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Shen, J., Jia, L., Dang, L. et al. StrucGP: de novo structural sequencing of site-specific N-glycan on glycoproteins using a modularization strategy. Nat Methods 18, 921–929 (2021). https://doi.org/10.1038/s41592-021-01209-0

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