( ...)Detection and Identification of Phosphorylation Sites in Proteins Using ,,, LC/MS/MS with Neutral Fragment Loss Mapping

Michaela Scigelova, Gary Woffendin; Thermo Finnigan, Hemel Hempstead, UK.
Malcolm Ward, Helen Byers; Proteome Sciences, London, UK.
Diane Hanger; Institute of Psychiatry KCL, London, UK.

Protein Phosphorylation

The data presented here can be acquired using any Thermo Finnigan LCQ Series ion trap mass spectrometer. Introduction Phosphorylation of proteins plays a pivotal role in the regulation of metabolic processes. Neurofilament proteins are important structural features of the neuronal cytoskeleton. Phosphorylation of these proteins is considered a critical factor for their assembly. Abnormal phosphorylation of neurofilaments is associated with some neurodegenerative conditions such as motor neuron disease, Parkinsons disease, and dementia.⁽¹⁾ Determination of endogenous phosphorylation sites is therefore important for understanding the role of neurofilament proteins in neurodegenerative disease.

Previously, the analysis of phosphorylation sites relied largely on conventional Edman sequencing following lengthy chromatographic separations or on two-dimensional phosphopeptide mapping of proteins radiolabelled with phosphate.⁽²⁾ More recently, a technique for the sequencing of proteins directly from polyacrylamide gels using electrospray in combination with tandem mass spectrometry has been developed. This allows the exclusive use of automated mass spectrometry techniques for sequencing and characterization of post-translational modifications from total digest mixtures. In this regime, proteins are uniquely identified by database searc
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