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Submitted on June 9, 2006
Revised on December 11, 2006
Accepted on January 4, 2007

Mitochondrial phosphoproteome revealed by an improved IMAC method and MS/MS/MS

Jaeick Lee, Yingda Xu, Yue Chen, Robert Sprung, Sung Chan Kim, Shanhai Xie, and Yingming Zhao

Biochemistry, UT Southwestern Medical Center, Dallas, TX 75390

Corresponding Author: yingming.zhao{at}utsouthwestern.edu

Immobilized metal ion affinity chromatography (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 32P-labeled tryptic peptides and development of MS/MS/MS (MS3) for identifying phosphopeptide sequences and phosphorylation sites. The improved IMAC method allows recovery of phosphorylated tryptic peptides up to ~77%, with only minor retention of unphosphorylated peptides. MS3 leads 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.


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