Proteomic Profiling pp 275-292

Part of the Methods in Molecular Biology book series (MIMB, volume 1295)

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

  • Marcella Nunes Melo-Braga
  • María Ibáñez-Vea
  • Martin Røssel Larsen
  • Katarzyna Kulej

Abstract

Post-translational modifications (PTMs) such as phosphorylation, acetylation, and glycosylation are an essential regulatory mechanism of protein function and they are associated with a 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 generally 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 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 most of the more than 300 known modifications we have none 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 is subsequently subjected to a next round of TiO2 enrichment for further separation of mono-phosphopeptides from deglycosylated peptides. In addition, the acetylated peptides present in the first TiO2 flow-through are enriched by immunoprecipitation (IP). Finally, the samples are fractionated by hydrophilic interaction liquid chromatography (HILIC) to reduce sample complexity and increase the coverage during 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.

Key words

Protein post-translational modification (PTM) enrichment Phosphorylation Acetylation Sialic acid (SA)-glycosylation Immunoprecipitation (IP) TiSH Comprising of titanium dioxide (TiO2), Sequential elution from immobilized metal affinity chromatography (SIMAC) and hydrophilic interaction liquid chromatography (HILIC) Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) 

References

  1. 1.
    Andersson L, Porath J (1986) Isolation of phosphoproteins by immobilized metal (Fe3+) affinity chromatography. Anal Biochem 154:250–254CrossRefPubMedGoogle Scholar
  2. 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 6:2436–2445CrossRefPubMedCentralPubMedGoogle Scholar
  3. 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 76:3935–3943CrossRefPubMedGoogle Scholar
  4. 4.
    Kuroda I, Shintani Y, Motokawa M, Abe S, Furuno M (2004) Phosphopeptide-selective column-switching RP-HPLC with a titania precolumn. Anal Sci 20:1313–1319CrossRefPubMedGoogle Scholar
  5. 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 4:873–886CrossRefPubMedGoogle Scholar
  6. 6.
    Bodenmiller B, Mueller LN, Mueller M et al (2007) Reproducible isolation of distinct, overlapping segments of the phosphoproteome. Nat Methods 4:231–237CrossRefPubMedGoogle Scholar
  7. 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 7:661–671CrossRefPubMedGoogle Scholar
  8. 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 Proteomics 75:5749–5761CrossRefPubMedGoogle Scholar
  9. 9.
    Jensen SS, Larsen MR (2007) Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques. Rapid Commun Mass Spectrom 21:3635–3645CrossRefPubMedGoogle Scholar
  10. 10.
    Larsen MR, Jensen SS, Jakobsen LA, Heegaard NH (2007) Exploring the sialiome using titanium dioxide chromatography and mass spectrometry. Mol Cell Proteomics 6:1778–1787CrossRefPubMedGoogle Scholar
  11. 11.
    Bunkenborg J, Pilch BJ, Podtelejnikov AV, Wisniewski JR (2004) Screening for N-glycosylated proteins by liquid chromatography mass spectrometry. Proteomics 4:454–465CrossRefPubMedGoogle Scholar
  12. 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 21:660–666CrossRefPubMedGoogle Scholar
  13. 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 5:1974–1982CrossRefPubMedGoogle Scholar
  14. 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 11:1191–202, M112.017509 [pii] 10.1074/mcp.M112.017509CrossRefPubMedCentralPubMedGoogle Scholar
  15. 15.
    Edwards AV, Schwammle V, Larsen MR (2014) Neuronal process structure and growth proteins are targets of heavy PTM regulation during brain development. J Proteomics 101:77–87CrossRefPubMedGoogle Scholar
  16. 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 13:311–328CrossRefPubMedCentralPubMedGoogle Scholar
  17. 17.
    Kim SC, Sprung R, Chen Y et al (2006) Substrate and functional diversity of lysine acetylation revealed by a proteomics survey. Mol Cell 23:607–618CrossRefPubMedGoogle Scholar
  18. 18.
    Zhang J, Sprung R, Pei J et al (2009) Lysine acetylation is a highly abundant and evolutionarily conserved modification in Escherichia coli. Mol Cell Proteomics 8:215–225CrossRefPubMedCentralPubMedGoogle Scholar
  19. 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 11:945–956CrossRefPubMedCentralPubMedGoogle Scholar
  20. 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 11:735–742CrossRefPubMedCentralPubMedGoogle Scholar
  21. 21.
    Choudhary C, Kumar C, Gnad F et al (2009) Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 325:834–840CrossRefPubMedGoogle Scholar
  22. 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. doi:10.1038/msb.2012.4 PubMedCentralPubMedGoogle Scholar
  23. 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 13:260–267CrossRefPubMedGoogle Scholar
  24. 24.
    Mertins P, Qiao JW, Patel J et al (2013) Integrated proteomic analysis of post-translational modifications by serial enrichment. Nat Methods 10:634–637CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Marcella Nunes Melo-Braga
    • 1
  • María Ibáñez-Vea
    • 1
  • Martin Røssel Larsen
    • 1
  • Katarzyna Kulej
    • 1
  1. 1.Department of Biochemistry and Molecular BiologyUniversity of Southern DenmarkOdense MDenmark

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