Skip to main content

Quantitative Phosphoproteomic Using Titanium Dioxide Micro-Columns and Label-Free Quantitation

Part of the Methods in Molecular Biology book series (MIMB,volume 1977)


Phosphorylation events are important during cellular function. Analysis of phosphorylation in complex samples has been extensively studied using large-scale phosphopeptide enrichment methods. Quantitative analysis of the enriched phosphopeptides is subsequently performed using label-based methodologies (e.g., SILAC, iTRAQ, and others). Here we describe the protocol for the quantitative analysis of phosphopeptides, enriched with titanium dioxide micro-column, using an intensity-based label-free quantitation.

Key words

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

Buying options

USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
USD   99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more


  1. Graves JD, Krebs EG (1999) Protein phosphorylation and signal transduction. Pharmacol Ther 82(2–3):111–121

    Article  CAS  Google Scholar 

  2. 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(7):873–886

    Article  CAS  Google Scholar 

  3. Thingholm TE, Jorgensen TJ, Jensen ON et al (2006) Highly selective enrichment of phosphorylated peptides using titanium dioxide. Nat Protoc 1(4):1929–1935

    Article  CAS  Google Scholar 

  4. Cantin GT, Yi W, Lu B et al (2008) Combining protein-based IMAC, peptide-based IMAC, and MudPIT for efficient phosphoproteomic analysis. J Proteome Res 7(3):1346–1351

    Article  CAS  Google Scholar 

  5. Ong SE, Blagoev B, Kratchmarova I et al (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1(5):376–386

    Article  CAS  Google Scholar 

  6. Altelaar AF, Frese CK, Preisinger C et al (2013) Benchmarking stable isotope labeling based quantitative proteomics. J Proteome 88:14–26

    Article  CAS  Google Scholar 

  7. Kwon OK, Kim SJ, Lee YM et al (2016) Global analysis of phosphoproteome dynamics in embryonic development of zebrafish (Danio rerio). Proteomics 16(1):136–149

    Article  CAS  Google Scholar 

  8. Cantin GT, Venable JD, Cociorva D et al (2006) Quantitative phosphoproteomic analysis of the tumor necrosis factor pathway. J Proteome Res 5(1):127–134

    Article  CAS  Google Scholar 

  9. Hu X, Wu L, Zhao F et al (2015) Phosphoproteomic analysis of the response of maize leaves to drought, heat and their combination stress. Front Plant Sci 6:298

    PubMed  PubMed Central  Google Scholar 

  10. Nguyen TH, Brechenmacher L, Aldrich JT et al (2012) Quantitative phosphoproteomic analysis of soybean root hairs inoculated with Bradyrhizobium japonicum. Mol Cell Proteomics 11(11):1140–1155

    Article  Google Scholar 

  11. Ma Q, Wu M, Pei W et al (2014) Quantitative phosphoproteomic profiling of fiber differentiation and initiation in a fiberless mutant of cotton. BMC Genomics 15:466

    Article  Google Scholar 

  12. Jensen SS, Larsen MR (2007) Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques. Rapid Commun Mass Spectrom 21(22):3635–3645

    Article  CAS  Google Scholar 

  13. Thingholm TE, Larsen MR (2009) The use of titanium dioxide micro-columns to selectively isolate phosphopeptides from proteolytic digests. Methods Mol Biol 527:57–66. xi

    Article  CAS  Google Scholar 

  14. Le Bihan T, Hindle M, Martin SF et al (2015) Label-free quantitative analysis of the casein kinase 2-responsive phosphoproteome of the marine minimal model species Ostreococcus tauri. Proteomics 15(23–24):4135–4144

    Article  Google Scholar 

  15. Hindle MM, Le Bihan T, Krahmer J et al (2016) qpMerge: merging different peptide isoforms using a motif centric strategy. bioRxiv 047100;

Download references

Author information

Authors and Affiliations


Corresponding author

Correspondence to Martin E. Barrios-Llerena .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Barrios-Llerena, M.E., Le Bihan, T. (2019). Quantitative Phosphoproteomic Using Titanium Dioxide Micro-Columns and Label-Free Quantitation. In: Evans, C., Wright, P., Noirel, J. (eds) Mass Spectrometry of Proteins. Methods in Molecular Biology, vol 1977. Humana Press, New York, NY.

Download citation

  • DOI:

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9231-7

  • Online ISBN: 978-1-4939-9232-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics