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Comprehensive Protocol to Simultaneously Study Protein Phosphorylation, Acetylation, and N-Linked Sialylated Glycosylation

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Proteomic Profiling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2261))

Abstract

Posttranslational modifications (PTMs) such as phosphorylation, acetylation, and glycosylation are an essential regulatory mechanism of protein function and interaction, and they are associated with a wide range of biological processes. Since most PTMs alter the molecular mass of a protein, mass spectrometry (MS) is the ideal analytical tool for studying various PTMs. However, PTMs are often present in substoichiometric levels, and therefore their unmodified counterpart often suppresses their signal in MS. Consequently, PTM analysis by MS is a challenging task, requiring highly specialized and sensitive PTM-specific enrichment methods. Currently, several methods have been implemented for PTM enrichment, and each of them has its drawbacks and advantages as they differ in selectivity and specificity toward specific protein modifications. Unfortunately, for the vast majority of more than 400 known modifications, we have no or poor tools for selective enrichment.

Here, we describe a comprehensive workflow to simultaneously study phosphorylation, acetylation, and N-linked sialylated glycosylation from the same biological sample. The protocol involves an initial titanium dioxide (TiO2) step to enrich for phosphopeptides and sialylated N-linked glycopeptides followed by glycan release and post-fractionation using sequential elution from immobilized metal affinity chromatography (SIMAC) to separate mono-phosphorylated and deglycosylated peptides from multi-phosphorylated ones. The IMAC flow-through and acidic elution are subsequently subjected to a next round of TiO2 enrichment for further separation of mono-phosphopeptides from deglycosylated peptides. Furthermore, the lysine-acetylated peptides present in the first TiO2 flow-through fraction are enriched by immunoprecipitation (IP) after peptide cleanup. Finally, the samples are fractionated by high pH reversed phase chromatography (HpH) or hydrophilic interaction liquid chromatography (HILIC ) to reduce sample complexity and increase the coverage in the subsequent LC-MS /MS analysis. This allows the analysis of multiple types of modifications from the same highly complex biological sample without decreasing the quality of each individual PTM study.

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References

  1. Andersson L, Porath J (1986) Isolation of phosphoproteins by immobilized metal (Fe3+) affinity chromatography. Anal Biochem 154:250–254

    Article  CAS  Google Scholar 

  2. Neville DC, Rozanas CR, Price EM, Gruis DB, Verkman AS, Townsend RR (1997) Evidence for phosphorylation of serine 753 in CFTR using a novel metal-ion affinity resin and matrix-assisted laser desorption mass spectrometry. Protein Sci 6:2436–2445

    Article  CAS  Google Scholar 

  3. Pinkse MW, Uitto PM, Hilhorst MJ et al (2004) Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns. Anal Chem 76:3935–3943

    Article  CAS  Google Scholar 

  4. Kuroda I, Shintani Y, Motokawa M, Abe S, Furuno M (2004) Phosphopeptide-selective column-switching RP-HPLC with a titania precolumn. Anal Sci 20:1313–1319

    Article  CAS  Google Scholar 

  5. Larsen MR, Thingholm TE, Jensen ON et al (2005) Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol Cell Proteomics 4:873–886

    Article  CAS  Google Scholar 

  6. Bodenmiller B, Mueller LN, Mueller M et al (2007) Reproducible isolation of distinct, overlapping segments of the phosphoproteome. Nat Methods 4:231–237

    Article  CAS  Google Scholar 

  7. Thingholm TE, Jensen ON, Robinson PJ, Larsen MR (2008) SIMAC (sequential elution from IMAC), a phosphoproteomics strategy for the rapid separation of monophosphorylated from multiply phosphorylated peptides. Mol Cell Proteomics 7:661–671

    Article  CAS  Google Scholar 

  8. Engholm-Keller K, Birck P, Storling J et al (2012) TiSH—a robust and sensitive global phosphoproteomics strategy employing a combination of TiO2, SIMAC, and HILIC. J Proteome 75:5749–5761

    Article  CAS  Google Scholar 

  9. Jensen SS, Larsen MR (2007) Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques. Rapid Commun Mass Spectrom 21:3635–3645

    Article  CAS  Google Scholar 

  10. Larsen MR, Jensen SS, Jakobsen LA, Heegaard NH (2007) Exploring the sialiome using titanium dioxide chromatography and mass spectrometry. Mol Cell Proteomics 6:1778–1787

    Article  CAS  Google Scholar 

  11. Bunkenborg J, Pilch BJ, Podtelejnikov AV, Wisniewski JR (2004) Screening for N-glycosylated proteins by liquid chromatography mass spectrometry. Proteomics 4:454–465

    Article  CAS  Google Scholar 

  12. Zhang H, Li XJ, Martin DB, Aebersold R (2003) Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Nat Biotechnol 21:660–666

    Article  CAS  Google Scholar 

  13. Palmisano G, Lendal SE, Engholm-Keller K et al (2010) Selective enrichment of sialic acid-containing glycopeptides using titanium dioxide chromatography with analysis by HILIC and mass spectrometry. Nat Protoc 5:1974–1982

    Article  CAS  Google Scholar 

  14. Palmisano G, Parker BL, Engholm-Keller K et al (2012) A novel method for the simultaneous enrichment, identification and quantification of phosphopeptides and sialylated glycopeptides applied to a temporal profile of mouse brain development. Mol Cell Proteomics. https://doi.org/10.1074/mcp.M112.017509

  15. Edwards AV, Schwammle V, Larsen MR (2014) Neuronal process structure and growth proteins are targets of heavy PTM regulation during brain development. J Proteome 101:77–87

    Article  CAS  Google Scholar 

  16. Melo-Braga MN, Schulz M, Liu Q et al (2013) Comprehensive quantitative comparison of the membrane proteome, phosphoproteome and sialiome of human embryonic and neural stem cells. Mol Cell Proteomics 13:311–328

    Article  Google Scholar 

  17. Kim SC, Sprung R, Chen Y et al (2006) Substrate and functional diversity of lysine acetylation revealed by a proteomics survey. Mol Cell 23:607–618

    Article  CAS  Google Scholar 

  18. Zhang J, Sprung R, Pei J, at al. (2009) Lysine acetylation is a highly abundant and evolutionarily conserved modification in Escherichia coli. Mol Cell Proteomics 8:215–225

    Article  CAS  Google Scholar 

  19. Melo-Braga MN, Verano-Braga T, Leon IR et al (2012) Modulation of protein phosphorylation, N-glycosylation and Lys-acetylation in grape (Vitis vinifera) mesocarp and exocarp owing to Lobesia botrana infection. Mol Cell Proteomics 11:945–956

    Article  CAS  Google Scholar 

  20. Jeffers V, Sullivan WJ Jr (2012) Lysine acetylation is widespread on proteins of diverse function and localization in the protozoan parasite Toxoplasma gondii. Eukaryot Cell 11:735–742

    Article  CAS  Google Scholar 

  21. Choudhary C, Kumar C, Gnad F et al (2009) Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 325:834–840

    Article  CAS  Google Scholar 

  22. van Noort V, Seebacher J, Bader S et al (2012) Cross-talk between phosphorylation and lysine acetylation in a genome-reduced bacterium. Mol Syst Biol 8:571. https://doi.org/10.1038/msb.2012.4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Edwards AV, Edwards GJ, Schwammle V et al (2014) Spatial and temporal effects in protein post-translational modification distributions in the developing mouse brain. J Proteome Res 13:260–267

    Article  CAS  Google Scholar 

  24. Aebersold R, Agar JN, Amster IJ, Baker MS, Bertozzi CR, Boja ES et al (2018) How many human proteoforms are there? Nat Chem Biol 14:206–214

    Article  CAS  Google Scholar 

  25. Mertins P, Qiao JW, Patel J et al (2013) Integrated proteomic analysis of post-translational modifications by serial enrichment. Nat Methods 10:634–637

    Article  CAS  Google Scholar 

  26. Parker BL, Shepherd NE, Trefely S, Hoffman NJ, White MY, Engholm-Keller K et al (2014) Structural basis for phosphorylation and lysine acetylation cross-talk in a kinase motif associated with myocardial ischemia and cardioprotection. J Biol Chem 289:25890–25906

    Article  CAS  Google Scholar 

  27. Engholm-Keller K, Birck P, Storling J, Pociot F, Mandrup-Poulsen T, Larsen MR (2012) TiSH—a robust and sensitive global phosphoproteomics strategy employing a combination of TiO2, SIMAC, and HILIC. J Proteome 75:5749–5761

    Article  CAS  Google Scholar 

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Acknowledgments

Marcella Nunes Melo-Braga, María Ibáñez-Vea, and Katarzyna Kulej contributed equally to this work.

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Author notes

  1. Marcella Nunes Melo-Braga, María Ibáñez-Vea and Katarzyna Kulej contributed equally with all other contributors.

Authors and Affiliations

  1. Department of Biochemistry and Immunology, Institute of Biological Science, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil

    Marcella Nunes Melo-Braga

  2. Genetics, Genomics and Microbiology Research Group, Health Science Department, Public University of Navarre, Pamplona, Navarra, Spain

    María Ibáñez-Vea

  3. Division of Protective Immunity and Division of Cancer Pathobiology, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA

    Katarzyna Kulej

  4. Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Katarzyna Kulej

  5. Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark

    Martin R. Larsen

Authors
  1. Marcella Nunes Melo-Braga
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  2. María Ibáñez-Vea
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  3. Katarzyna Kulej
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  4. Martin R. Larsen
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Corresponding author

Correspondence to Martin R. Larsen .

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Editors and Affiliations

  1. Bio-Rad Laboratories GmbH, Feldkirchen, Germany

    Anton Posch

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Melo-Braga, M.N., Ibáñez-Vea, M., Kulej, K., Larsen, M.R. (2021). Comprehensive Protocol to Simultaneously Study Protein Phosphorylation, Acetylation, and N-Linked Sialylated Glycosylation. In: Posch, A. (eds) Proteomic Profiling. Methods in Molecular Biology, vol 2261. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1186-9_5

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  • DOI: https://doi.org/10.1007/978-1-0716-1186-9_5

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1185-2

  • Online ISBN: 978-1-0716-1186-9

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