title: Quantitative analysis of global phosphorylation changes with high-resolution tandem mass spectrometry and stable isotopic labeling.
abstract: Quantitative measurement of specific protein phosphorylation sites is a primary interest of biologists, as site-specific phosphorylation information provides insights into cell signaling networks and cellular dynamics at a system level. Over the last decade, selective phosphopeptide enrichment methods including IMAC and metal oxides (TiO₂ and ZrO₂) have been developed and greatly facilitate large scale phosphoproteome analysis of various cells, tissues and living organisms, in combination with modern mass spectrometers featuring high mass accuracy and high mass resolution. Various quantification strategies have been applied to detecting relative changes in expression of proteins, peptides, and specific modifications between samples. The combination of mass spectrometry-based phosphoproteome analysis with quantification strategies provides a straightforward and unbiased method to identify and quantify site-specific phosphorylation. We describe common strategies for mass spectrometric analysis of stable isotope labeled samples, as well as two widely applied phosphopeptide enrichment methods based on IMAC(NTA-Fe³⁺) and metal oxide (ZrO₂). Instrumental configurations for on-line LC-tandem mass spectrometric analysis and parameters of conventional bioinformatic analysis of large data sets are also considered for confident identification, localization, and reliable quantification of site-specific phosphorylation.
authors.affiliation: Department of Biological Chemistry, University of Michigan, USA. hkweon@umich.edu
authors.forename: Hye Kyong
authors.initials: HK
authors.lastname: Kweon
authors.forename: Philip C
authors.initials: PC
authors.lastname: Andrews
mesh_terms: D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D006801:Humans; D007553:Isotope Labeling; D009571:Nitrilotriacetic Acid / Q000737:chemistry; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification*; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification*; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry; D015040:Zirconium / Q000737:chemistry
chemical_list: D010748:Phosphopeptides; D010750:Phosphoproteins; D015040:Zirconium; D009571:Nitrilotriacetic Acid; C028541:zirconium oxide