Isotope-labeling and affinity enrichment of phosphopeptides for proteomic analysis using liquid chromatography-tandem mass spectrometry.
The reversible phosphorylation of proteins is a dynamic process that plays a major role in many vital physiological processes by transmitting signals within cellular pathways and networks. Proteomic measurements using mass spectrometry are capable of characterizing the sites of protein phosphorylation and to quantify their change in abundance. However, the low stoichiometry of protein phosphorylation events often preclude mass spectrometry detection and require additional sample preparation steps to facilitate their characterization. Many analytical methods have been used to map and quantify changes in phosphorylation, and this chapter will present two methods that can be used for extraction of phosphopeptides from protein and proteome digests to map phosphorylation sites using liquid chromatography-tandem mass spectrometry (LC/MS/MS). The first method describes an immobilized metal affinity chromatography (IMAC) technique using Ga3+ to enrich for phosphopeptides from protein digests. The second method describes the utilization of phosphoprotein isotope-coded solid-phase tags (PhIST) to label and enrich phosphopeptides from complex mixtures to both identify and quantify changes in protein phosphorylation. The IMAC and PhIST protocols can be applied to any isolated protein sample and is amenable to additional fractionation using strong cation/anion exchange chromatography prior to reversed-phase LC/MS/MS analysis.
Kota
Uma
U
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695-7622, USA.
Chien
Ko-yi
KY
Goshe
Michael B
MB
D002846:Chromatography, Affinity / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007553:Isotope Labeling; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000032:analysis*; D010766:Phosphorylation; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D010748:Phosphopeptides; D020543:Proteome