Phosphorylation. site analysis by mass spectrometry

Abstract

Abstract The determination of phosphorylation sites on proteins is a vital part of their characterization, and an important step in understanding the structural basis of how phosphorylation modulates their functions. With the recent explosive growth in the use of mass spectrometry in the structural analysis of proteins and peptides, this approach has also become the method of choice for the elucidation of post-translational modifications, including phosphorylation. Many developments have taken place since early studies using fast-atom bombardment mass spectrometry (FABMS) in 1985 (1). Matrix-assisted laser desorption/ionization time-of-flight (MALDITOF), electrospray ionization (ESI) triple quadrupole mass spectrometry and its low flow rate version, nano-ESI mass spectrometry, are currently the most widely used forms of instrumentation in these studies. They are both sensitive and highly precise, and this chapter will be confined to these instrumentation types, with which we have practical experience. Hybrid instruments e.g. Q-TOF (quadrupole time-of-flight) are now available, or in advanced stages of development, from several manufacturers, as well as both electrospray and MALDITOF instruments with Fourier transform detection at the frontiers of precision. Other mass spectrometric analytical methodologies, including ion-trap methods, have been applied to phosphorylation site analysis. It is now essential for all laboratories involved in protein phosphorylation studies to have access to mass spectrometry if they are to contribute to understanding this pre-eminent regulatory mechanism.