Analytical Strategies in Mass Spectrometry-Based Phosphoproteomics

  • Heidi Rosenqvist
  • Juanying Ye
  • Ole N. JensenEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 753)


Phosphoproteomics, the systematic study of protein phosphorylation events and cell signaling networks in cells and tissues, is a rapidly evolving branch of functional proteomics. Current phosphoproteomics research provides a large toolbox of strategies and protocols that may assist researchers to reveal key regulatory events and phosphorylation-mediated processes in the cell and in whole organisms. We present an overview of sensitive and robust analytical methods for phosphopeptide analysis, including calcium phosphate precipitation and affinity enrichment methods such as IMAC and TiO2. We then discuss various tandem mass spectrometry approaches for phosphopeptide sequencing and quantification, and we consider aspects of phosphoproteome data analysis and interpretation. Efficient integration of these stages of phosphoproteome analysis is highly important to ensure a successful outcome of large-scale experiments for studies of phosphorylation-mediated protein regulation.

Key words

Protein phosphorylation cell signaling IMAC TiO2 tandem mass spectrometry, phosphopeptide sequencing 



Research in O.N.J.’s laboratory is funded by grants from the Danish Research Councils, The Danish National Research Foundation, and The Lundbeck Foundation. H.R. was funded by a Ph.D. fellowship from the German Research Council (DFG, grant number WI 2044/5-1).


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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular BiologyUniversity of Southern DenmarkOdenseDenmark
  2. 2.Strathclyde Institute of Pharmacy and Biomedical SciencesUniversity of StrathclydeScotlandUK

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