Mitochondrial Phosphoproteome Revealed by an Improved IMAC Method and MS/MS/MS

Abstract

Global analysis of protein phosphorylation will provide insight into mechanisms by which this dynamic post-translational modification modulates diverse cellular processes. Mass spectrometry-based phosphoproteomics is a potentially powerful approach for global profiling and quantification of protein phosphorylation. Such studies usually involve selective isolation of phosphorylated peptides and their subsequent fragmentation in a mass spectrometer to assign the sequence and localize phosphorylation sites. Three strategies have been described for enriching phosphopeptides based on antibodies (1, 2), chemical derivatization (3, 4), or ionic interactions (e.g. IMAC and strong ion exchange chromatography) (5-9). These methods have achieved limited success in enriching phosphopeptides for proteomics studies. Among these approaches, IMAC is the most convenient and holds much potential for the efficient isolation of phosphopeptides (10 -13). The method has been used for several global analyses of protein phosphorylation in model organisms and cellular organelles (5-9). Nevertheless further refinement of extant IMAC protocols is required to achieve high reproducibility and efficiency (14).Poor fragmentation of phosphopeptides in the mass spectrometer represents the second major challenge for phosphoproteomics. The availability of a relatively low energy fragmentation pathway via ␤-elimination of the phosphate moiety limits fragmentation at peptide bonds that would be informative for identifying the sequence and site(s) of phosphorylation of the peptide. Mass spectrometers under development, such as instruments using electron transfer dissociation (15), might address this problem. However, such mass spectrometers are not currently commercially available. In addition, fragmentation of doubly charged and singly charged peptides in electron transfer dissociation mass spectrometry is compromised. In summary, despite extensive effort in the past several years, efficient proteomics of protein phosphorylation remains a daunting challenge.Here we report analysis of the phosphoproteome of mitochondria using an improved method that integrates an optimized batchwise IMAC protocol for isolation of phosphopeptides and MS3 1 for fragmentation of phosphopeptides. The improved IMAC procedure allowed recovery of ϳ77% of phosphopeptides while retaining few unphosphorylated peptides. MS3 addressed the poor fragmentation of phosphopeptides by generating highly informative fragmentation patterns that allow peptide identification. Analysis of mitochondrial phosphorylation revealed 84 phosphorylation sites from 62 proteins. Most identified phosphorylation sites have not been reported before, providing novel information about mitochondrial regulatory mechanisms. The optimized batchwise IMAC protocol in combination with MS3 offers a relatively simple and more efficient approach for proteomics of protein phosphorylation.EXPERIMENTAL PROCEDURES

Evolutionary Constraints of Phosphorylation in Eukaryotes, Prokaryotes, and Mitochondria

“…The same pattern was observed for phosphorylated proteins. Overall, 28% of the phosphorylated mitochondrial proteins (from our set as well as from the Lee et al (29) data set) are conserved in a prokaryotic species compared with 20% of the phosphorylated non-mitochondrial proteins. These observations are in concordance with the prokaryotic character of mitochondria expected from the endosymbiotic hypothesis.…”

ArMone: A Software Suite Specially Designed for Processing and Analysis of Phosphoproteome Data

“…It has become one of the most commonly used tools for high through-put analysis of protein phosphorylation. [4][5][6][7][8][9][10][11] As most of the database search engines, especially the free search engines, do not provide user-friendly pre-and postsearch data processing tools, various proteome pipelines have been developed. [12][13][14][15] These pipelines commonly contain modules with various functions such as validation of peptide and protein identification, quantitative analysis, and so on.…”

Facile preparation of monolithic immobilized metal affinity chromatography capillary columns for selective enrichment of phosphopeptides

“…Slurries of Ti 41 -IMAC and Zr 41 -IMAC beads were prepared as described previously [27], with minor modifications. Briefly, IMAC beads (20 mg) were weighed into a 1.5-mL microcentrifuge tube.…”