Protein Protocols Protein Protocols

Mass Spectrometry of Protein Phosphorylation 609
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Mass Spectrometric Analysis of Protein Phosphorylation
Débora Bonenfant, Thierry Mini, and Paul Jenö
1. Introduction
Phosphorylation is one the most frequently occurring posttranslational modifications
in proteins, playing an essential role in transferring signals from the outside to the
inside of a cell and in regulating many diverse cellular processes such as growth,
metabolism, proliferation, motility, and differentiation. It is estimated that up to one
third of all proteins in a typical mammalian cell are phosphorylated (1). Phosphorylation
is carried out by a vast group of protein kinases which are thought to constitute 3%
of the entire eukaryotic genome (1–3). To decipher the recognition signal of protein
kinases and protein phosphatases acting on a given molecular target, and to understand
how the activity of the target protein is regulated by phosphorylation, it is important to
define the sites and the extent of phosphorylation at each specific site.
The most commonly employed technique of phosphoprotein analysis involves in
vivo or in vitro labeling with [ 32P]phosphate (4–8). The radiolabeled protein is subsequently
digested with a suitable protease and radiolabeled peptides are separated either
by high-performance liquid chromatography (HPLC) (7,8) or by two-dimensional
phosphopeptide mapping (4–8). The site of phosphorylation is then determined by
solid-phase Edman sequencing (6).
Identification of phosphorylation sites by Edman degradation of [ 32P]-labeled proteins
remains a valuable approach when working with limited quantities of protein. In
the last couple of years, however, electrospray ionization-mass spectrometry (ESI-MS)
and matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) have
been used quite successfully for phosphoprotein analysis (9–12). Mass spectrometry is
particularly useful when radioactive protein labeling cannot be performed, when phosphorylation
sites are clustered within a short peptide sequence, or when the sites of phosphorylation
are located more than 10–15 residues from the amino-(N)-terminus of a peptide.
The basic elements of mass spectrometric phosphoprotein analysis consist of proteolytic
digestion of the protein of interest followed by measuring the masses of the
resulting peptides. When the protein sequence is known, peptides are identified by
comparing observed masses to those predicted based on the specificity of the protease.
Phosphopeptides have masses 80 Da greater (due to the presence of an HPO3 group)
than predicted from the peptide sequence; hence masses increasing in multiples of 80 Da
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
609