title: Mitochondrial phosphoproteome revealed by an improved IMAC method and MS/MS/MS.
abstract: IMAC in combination with mass spectrometry is a promising approach for global analysis of protein phosphorylation. Nevertheless this approach suffers from two shortcomings: inadequate efficiency of IMAC and poor fragmentation of phosphopeptides in the mass spectrometer. Here we report optimization of the IMAC procedure using (32)P-labeled tryptic peptides and development of MS/MS/MS (MS3) for identifying phosphopeptide sequences and phosphorylation sites. The improved IMAC method allowed recovery of phosphorylated tryptic peptides up to approximately 77% with only minor retention of unphosphorylated peptides. MS3 led to efficient fragmentation of the peptide backbone in phosphopeptides for sequence assignment. Proteomics of mitochondrial phosphoproteins using the resulting IMAC protocol and MS3 revealed 84 phosphorylation sites in 62 proteins, most of which have not been reported before. These results revealed diverse phosphorylation pathways involved in the regulation of mitochondrial functions. Integration of the optimized batchwise IMAC protocol with MS3 offers a relatively simple and more efficient approach for proteomics of protein phosphorylation.
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9038, USA.
authors.forename: Jaeick
authors.initials: J
authors.lastname: Lee
authors.forename: Yingda
authors.initials: Y
authors.lastname: Xu
authors.forename: Yue
authors.initials: Y
authors.lastname: Chen
authors.forename: Robert
authors.initials: R
authors.lastname: Sprung
authors.forename: Sung Chan
authors.initials: SC
authors.lastname: Kim
authors.forename: Shanhai
authors.initials: S
authors.lastname: Xie
authors.forename: Yingming
authors.initials: Y
authors.lastname: Zhao
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D051379:Mice; D008930:Mitochondria, Liver / Q000737:chemistry; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000302:isolation & purification*; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification*; D010766:Phosphorylation; D020543:Proteome / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D024101:Mitochondrial Proteins; D010750:Phosphoproteins; D020543:Proteome