Skip to main content

Advertisement

Log in

Isotope-targeted glycoproteomics (IsoTaG) analysis of sialylated N- and O-glycopeptides on an Orbitrap Fusion Tribrid using azido and alkynyl sugars

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Protein glycosylation is a post-translational modification (PTM) responsible for many aspects of proteomic diversity and biological regulation. Assignment of intact glycan structures to specific protein attachment sites is a critical step towards elucidating the function encoded in the glycome. Previously, we developed isotope-targeted glycoproteomics (IsoTaG) as a mass-independent mass spectrometry method to characterize azide-labeled intact glycopeptides from complex proteomes. Here, we extend the IsoTaG approach with the use of alkynyl sugars as metabolic labels and employ new probes in analysis of the sialylated glycoproteome from PC-3 cells. Using an Orbitrap Fusion Tribrid mass spectrometer, we identified 699 intact glycopeptides from 192 glycoproteins. These intact glycopeptides represent a total of eight sialylated glycan structures across 126 N- and 576 O-glycopeptides. IsoTaG is therefore an effective platform for identification of intact glycopeptides labeled by alkynyl or azido sugars and will facilitate further studies of the glycoproteome.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Amon R, Reuven EM, Leviatan Ben-Arye S, Padler-Karavani V. Glycans in immune recognition and response. Carbohydr Res. 2014;389:115.

    Article  CAS  Google Scholar 

  2. Adamczyk B, Tharmalingam T, Rudd PM. Glycans as cancer biomarkers. Biochim Biophys Acta. 2012;1820(9):1347.

    Article  CAS  Google Scholar 

  3. Song E, Hu Y, Hussein A, Yu C-Y, Tang H, Mechref Y. Characterization of the glycosylation site of human PSA prompted by missense mutation using LC–MS/MS. J Proteome Res. 2015;14(7):2872.

    Article  CAS  Google Scholar 

  4. Paszek MJ, DuFort CC, Rossier O, Bainer R, Mouw JK, Godula K, et al. The cancer glycocalyx mechanically primes integrin-mediated growth and survival. Nature. 2014;511:319.

    Article  CAS  Google Scholar 

  5. Brown JR, Fuster MM, Li R, Varki N, Glass CA, Esko JD. A disaccharide-based inhibitor of glycosylation attenuates metastatic tumor cell dissemination. Clin Cancer Res. 2006;12(9):2894.

    Article  CAS  Google Scholar 

  6. Samraj AN, Pearce OMT, Läubli H, Crittenden AN, Bergfeld AK, Banda K, et al. A red meat-derived glycan promotes inflammation and cancer progression. Proc Natl Acad Sci U S A. 2015;112(2):542.

    Article  CAS  Google Scholar 

  7. Hudak JE, Canham SM, Bertozzi CR. Glycocalyx engineering reveals a Siglec-based mechanism for NK cell immunoevasion. Nat Chem Biol. 2014;10(1):69.

    Article  CAS  Google Scholar 

  8. Xiao H, Woods EC, Vukojicic P, Bertozzi CR, Precision glycocalyx editing as a strategy for cancer immunotherapy. Proc Natl Acad Sci in press, 2016.

  9. Miyoshi E, Moriwaki K, Nakagawa T. Biological function of fucosylation in cancer biology. J Biochem. 2008;143(6):725.

    Article  CAS  Google Scholar 

  10. Okeley NM, Alley SC, Anderson ME, Boursalian TE, Burke PJ, Emmerton KM, et al. Development of orally active inhibitors of protein and cellular fucosylation. Proc Natl Acad Sci U S A. 2013;110(14):5404.

    Article  CAS  Google Scholar 

  11. Liener I, The lectins: properties, functions, and applications in biology and medicine. Elsevier, 1986.

  12. Belardi B, Bertozzi CR. Chemical lectinology: Tools for probing the ligands and dynamics of mammalian lectins in vivo. Chem Biol. 2015;22(8):983.

    Article  CAS  Google Scholar 

  13. Brooks SA. Strategies for analysis of the glycosylation of proteins: current status and future perspectives. Mol Biotechnol. 2009;43(1):76.

    Article  CAS  Google Scholar 

  14. Chen C-C, Su W-C, Huang B-Y, Chen Y-J, Tai H-C, Obena RP. Interaction modes and approaches to glycopeptide and glycoprotein enrichment. Analyst (Cambridge, U K). 2014;139(4):688.

    Article  CAS  Google Scholar 

  15. Zhang H, Li XJ, Martin DB, Aebersold R. Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Nat Biotechnol. 2003;21(6):660.

    Article  CAS  Google Scholar 

  16. Vosseller K, Trinidad JC, Chalkley RJ, Specht CG, Thalhammer A, Lynn AJ, et al. O-linked N-acetylglucosamine proteomics of postsynaptic density preparations using lectin weak affinity chromatography and mass spectrometry. Mol Cell Proteomics. 2006;5(5):923.

    Article  CAS  Google Scholar 

  17. Clark PM, Dweck JF, Mason DE, Hart CR, Buck SB, Peters EC, et al. Direct in-gel fluorescence detection and cellular imaging of O-GlcNAc-modified proteins. J Am Chem Soc. 2008;130(35):11576.

    Article  CAS  Google Scholar 

  18. Saxon E, Bertozzi CR. Cell surface engineering by a modified Staudinger reaction. Science. 2000;287(5460):2007.

    Article  CAS  Google Scholar 

  19. Thaysen-Andersen M, Packer NH, Schulz BL. Maturing glycoproteomics technologies provide unique structural insights into the N-glycoproteome and its regulation in health and disease. Mol Cell Proteomics. 2016;15(6):1773.

    Article  CAS  Google Scholar 

  20. Chandler KB, Costello CE. Glycomics and glycoproteomics of membrane proteins and cell-surface receptors: present trends and future opportunities. Electrophoresis. 2016;37(11):1407.

    Article  CAS  Google Scholar 

  21. Tarentino AL, Gomez CM, Plummer TH. Deglycosylation of asparagine-linked glycans by peptide:N-glycosidase F. Biochemistry. 1985;24(17):4665.

    Article  CAS  Google Scholar 

  22. Zielinska DF, Gnad F, Wiśniewski JR, Mann M. Precision mapping of an in vivo N-glycoproteome reveals rigid topological and sequence constraints. Cell. 2010;141(5):897.

    Article  CAS  Google Scholar 

  23. Song X, Ju H, Lasanajak Y, Kudelka MR, Smith DF, Cummings RD. Oxidative release of natural glycans for functional glycomics. Nat Meth. 2016;13(6):528.

    Article  CAS  Google Scholar 

  24. Alfaro JF, Gong C-X, Monroe ME, Aldrich JT, Clauss TRW, Purvine SO, et al. Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets. Proc Natl Acad Sci U S A. 2012;109(19):7280.

    Article  CAS  Google Scholar 

  25. Nilsson J, Ruetschi U, Halim A, Hesse C, Carlsohn E, Brinkmalm G, et al. Enrichment of glycopeptides for glycan structure and attachment site identification. Nat Meth. 2009;6(11):809.

    Article  CAS  Google Scholar 

  26. Steentoft C, Vakhrushev SY, Vester-Christensen MB, Schjoldager KT, Kong Y, Bennett EP, et al. Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines. Nat Meth. 2011;8(11):977.

    Article  CAS  Google Scholar 

  27. Steentoft C, Vakhrushev SY, Joshi HJ, Kong Y, Vester-Christensen MB, Schjoldager KT, et al. Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology. EMBO J. 2013;32(10):1478.

    Article  CAS  Google Scholar 

  28. Zhao P, Viner R, Teo CF, Boons G-J, Horn D, Wells L. Combining high-energy C-trap dissociation and electron transfer dissociation for protein O-GlcNAc modification site assignment. J Proteome Res. 2011;10(9):4088.

    Article  CAS  Google Scholar 

  29. Wu SW, Pu TH, Viner R, Khoo KH. Novel LC-MS(2) product dependent parallel data acquisition function and data analysis workflow for sequencing and identification of intact glycopeptides. Anal Chem. 2014;86(11):5478.

    Article  CAS  Google Scholar 

  30. Shah P, Wang X, Yang W, Toghi Eshghi S, Sun S, Hoti N, et al. Integrated proteomic and glycoproteomic analyses of prostate cancer cells reveal glycoprotein alteration in protein abundance and glycosylation. Mol Cell Proteomics. 2015;14(10):2753.

    Article  CAS  Google Scholar 

  31. Singh C, Zampronio CG, Creese AJ, Cooper HJ. Higher energy collision dissociation (HCD) product ion-triggered electron transfer dissociation (ETD) mass spectrometry for the analysis of N-linked glycoproteins. J Proteome Res. 2012;11(9):4517.

    Article  CAS  Google Scholar 

  32. Parker BL, Thaysen-Andersen M, Solis N, Scott NE, Larsen MR, Graham ME, et al. Site-specific glycan-peptide analysis for determination of N-glycoproteome heterogeneity. J Proteome Res. 2013;12(12):5791.

    Article  CAS  Google Scholar 

  33. He L, Xin L, Shan B, Lajoie GA, Ma B. GlycoMaster DB: software to assist the automated identification of N-linked glycopeptides by tandem mass spectrometry. J Proteome Res. 2014;13(9):3881.

    Article  CAS  Google Scholar 

  34. Hua S, Nwosu CC, Strum JS, Seipert RR, An HJ, Zivkovic AM, et al. Site-specific protein glycosylation analysis with glycan isomer differentiation. Anal Bioanal Chem. 2012;403(5):1291.

    Article  CAS  Google Scholar 

  35. Toghi Eshghi S, Shah P, Yang W, Li X, Zhang H. GPQuest: a spectral library matching algorithm for site-specific assignment of tandem mass spectra to intact N-glycopeptides. Anal Chem. 2015;87(10):5181.

    Article  CAS  Google Scholar 

  36. Woo CM, Iavarone AT, Spiciarich DR, Palaniappan KK, Bertozzi CR. Isotope-targeted glycoproteomics (IsoTaG): a mass-independent platform for intact N- and O-glycopeptide discovery and analysis. Nat Meth. 2015;12(6):561.

    Article  CAS  Google Scholar 

  37. Laughlin ST, Baskin JM, Amacher SL, Bertozzi CR. In vivo imaging of membrane-associated glycans in developing zebrafish. Science. 2008;320:664.

    Article  CAS  Google Scholar 

  38. Chang PV, Chen X, Smyrniotis C, Xenakis A, Hu T, Bertozzi CR, et al. Metabolic labeling of sialic acids in living animals with alkynyl sugars. Angew Chem Int Ed. 2009;48(22):4030.

    Article  CAS  Google Scholar 

  39. Hubbard SC, Boyce M, McVaugh CT, Peehl DM, Bertozzi CR. Cell surface glycoproteomic analysis of prostate cancer-derived PC-3 cells. Bioorg Med Chem Lett. 2011;21:4945.

    Article  CAS  Google Scholar 

  40. Hsu TL, Hanson SR, Kishikawa K, Wang SK, Sawa M, Wong CH. Alkynyl sugar analogs for the labeling and visualization of glycoconjugates in cells. Proc Natl Acad Sci U S A. 2007;104(8):2614.

    Article  CAS  Google Scholar 

  41. Chuh KN, Zaro BW, Piller F, Piller V, Pratt MR. Changes in metabolic chemical reporter structure yield a selective probe of O-GlcNAc modification. J Am Chem Soc. 2014;136(35):12283.

    Article  CAS  Google Scholar 

  42. Pangborn AB, Giardello MA, Grubbs RH, Rosen RK, Timmers FJ. Safe and convenient procedure for solvent purification. Organometallics. 1996;15(5):1518.

    Article  CAS  Google Scholar 

  43. Lee PJJ, Compton BJ, Destructible surfactants and uses thereof. USA Patent 2007.

  44. Wang W, Hong S, Tran A, Jiang H, Triano R, Liu Y, et al. Sulfated ligands for the copper(I)-catalyzed azide–alkyne cycloaddition. Chem Asian J. 2011;6(10):2796.

    Article  CAS  Google Scholar 

  45. Prescher JA, Dube DH, Bertozzi CR. Chemical remodelling of cell surfaces in living animals. Nature. 2004;430:873.

    Article  CAS  Google Scholar 

  46. Bern M, Kil YJ, Becker C, Byonic: advanced peptide and protein identification software. Curr Protoc Bioinformatics Chapter 13:Unit13 20, 2012.

  47. Vizcaino JA, Csordas A, del Toro N, Dianes JA, Griss J, Lavidas I, et al. 2016 update of the PRIDE database and its related tools. Nucleic Acids Res. 2016;44(D1):D447.

    Article  Google Scholar 

  48. Szychowski J, Mahdavi A, Hodas JJL, Bagert JD, Ngo JT, Landgraf P, et al. Cleavable biotin probes for labeling of biomolecules via azide–alkyne cycloaddition. J Am Chem Soc. 2010;132:18351.

    Article  CAS  Google Scholar 

  49. Palaniappan KK, Pitcher AA, Smart BP, Spiciarich DR, Iavarone AT, Bertozzi CR. Isotopic signature transfer and mass pattern prediction (IsoStamp): an enabling technique for chemically-directed proteomics. ACS Chem Biol. 2011;6(8):829.

    Article  CAS  Google Scholar 

  50. Woo CM, Bertozzi CR. Isotope targeted glycoproteomics (IsoTaG) to characterize intact, metabolically labeled glycopeptides from complex proteomes. Curr Protoc Chem Biol. 2016;8(1):59.

    Article  Google Scholar 

Download references

Acknowledgments

Financial support from the US National Institutes of Health (CA200423, C.R.B.), Jane Coffin Childs Memorial Fund (C.M.W.), Burroughs Wellcome Fund Career Awards at the Scientific Interface (C.M.W.), Stanford Undergraduate Advising and Research Student Grant (A.F.), the W.M. Keck Foundation Medical Research Program (J.E.E.), and the Bill and Melinda Gates Foundation (J.E.E.) are gratefully acknowledged.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Carolyn R. Bertozzi.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Published in the topical collection Glycomics, Glycoproteomics and Allied Topics with guest editors Yehia Mechref and David Muddiman.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 3393 kb)

ESM 2

(XLSX 571 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Woo, C.M., Felix, A., Zhang, L. et al. Isotope-targeted glycoproteomics (IsoTaG) analysis of sialylated N- and O-glycopeptides on an Orbitrap Fusion Tribrid using azido and alkynyl sugars. Anal Bioanal Chem 409, 579–588 (2017). https://doi.org/10.1007/s00216-016-9934-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-016-9934-9

Keywords

Navigation