Application of immobilized metal affinity chromatography and mass spectrometry towards the identification of protein phosphorylation: Finding the needle in the haystack

Mass spectrometry has emerged as the technique of choice for the identification of protein phosphorylation due to it being a high throughput, high sensitivity technique. Phosphorylated peptides were enriched by immobilized metal affinity chromatography (IMAC) and identified by reversed phase liquid chromatography electrospray ionization mass spectrometry. These techniques in combination have led to the identification of numerous novel phosphorylation sites from samples as simple as purified single proteins to whole cell lysates. This dissertation will first present work that I have done to improve the technique of IMAC resulting in reproducible recovery of phosphopeptides at greater than 80%. The validity of the technique is demonstrated in Chapters 2 and 3 when the technique was applied to real life biological samples. In Chapter 2, twenty-eight phosphorylation sites on the cell migration protein talin were identified and then the phosphorylation levels were compared by stable isotope labeling of talin from resting and stimulated platelets. Two phosphorylation sites were identified as enhanced upon platelet stimulation to form clots. In Chapter 3, twelve phosphorylation sites were identified on the protein Nap1. The phosphorylation of Nap1 is believed to be cell cycle regulated and to control the function of Nap1 in histone import and mitotic progression. To better understand the pleiotropic functions of Nap1, the Nap1 interacting proteins were also identified by mass spectrometry.