Identification of phosphorylation sites using microimmobilized metal affinity chromatography

One of the most important roles that mass spectrometry (MS) has played in the late twentieth and early twenty-first centuries has been to assist in the growth of knowledge of dynamic phosphorylation events. Not only has MS allowed researches to pinpoint the site of phosphorylation, but it has also enabled them to identify the kinase/phosphatase pairs responsible for regulation of a specific modification as well as to follow the functional consequences of the observed phosphorylation events on the biology of the system. For phosphorylation analysis, the important contribution of MS has been critical but not definitive. There are numerous methods that have been applied with success, yet none are generally applicable to all analyses. So, for the time being, researchers in the field must select from a panel of methods to find (de)phosphorylation events. In the work described in this chapter, a collection of integrated methods are presented. A detailed account is provided for phosphorylation capture via on- and off-line immobilized metal affinity chromatography (IMAC). This is followed by a suite of useful strategies for discovery of phosphorylation positioning through sequence determination by phosphate-specific diagnostic ion scans, including precursor and product ion scans, neutral loss scans, and in-source dissociation and post-source decay.