Evaluation of HCD- and CID-type fragmentation within their respective detection platforms for murine phosphoproteomics.
ABSTRACT Protein phosphorylation modulates a myriad of biological functions, and its regulation is vital for proper cellular activity. Mass spectrometry is the enabling tool for phosphopeptide analysis, where recent instrumentation advances in both speed and sensitivity in linear ion trap and orbitrap technologies may yield more comprehensive phosphoproteomic analyses in less time. Protein phosphorylation analysis by MS relies on structural information derived through controlled peptide fragmentation. Compared with traditional, ion-trap-based collision-induced dissociation (CID), a more recent type of fragmentation termed HCD (higher energy collisional dissociation) provides beam type CID tandem MS with detection of fragment ions at high resolution in the orbitrap mass analyzer. Here we compared HCD to traditional CID for large-scale phosphorylation analyses of murine brain under three separate experimental conditions. These included a same-precursor analysis where CID and HCD scans were performed back-to-back, separate analyses of a phosphotyrosine peptide immunoprecipitation experiment, and separate whole phosphoproteome analyses. HCD generally provided higher search engine scores with more peptides identified, thus out-performing CID for back-to-back experiments for most metrics tested. However, for phosphotyrosine IPs and in a full phosphoproteome study of mouse brain, the greater acquisition speed of CID-only analyses provided larger data sets. We reconciled our results with those in direct contradiction from Nagaraj N, D'Souza RCJ et al. (J. Proteome Res. 9:6786, 2010). We conclude, for large-scale phosphoproteomics, CID fragmentation with rapid detection in the ion trap still produced substantially richer data sets, but the back-to-back experiments demonstrated the promise of HCD and orbitrap detection for the future.
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ABSTRACT: Phosphorylated O-GlcNAc is a novel post-translational modification that has so far only been found on the neuronal protein AP180 from the rat (Graham et al., J. Proteome Res. 2011, 10, 2725-2733). Upon collision induced dissociation, the modification generates a highly mass deficient fragment ion (m/z 284.0530) that can be used as a reporter for the identification of phosphorylated O-GlcNAc. Using a publically available mouse brain phosphoproteome data set, we employed our recently developed Oscore software to re-evaluate high resolution/high accuracy tandem mass spectra and discovered the modification on 23 peptides corresponding to 11 mouse proteins. The systematic analysis of 220 candidate phosphoGlcNAc tandem mass spectra as well as a synthetic standard enabled the dissection of the major phosphoGlcNAc fragmentation pathways, suggesting that the modification is O-GlcNAc-6-phosphate. We find that the classical O-GlcNAc modification often exists on the same peptides indicating that O-GlcNAc-6-phosphate may biosynthetically arise in two steps involving the O-GlcNAc transferase and a currently unknown kinase. Many of the identified proteins are involved in synaptic transmission and for Ca(2+)/calmodulin kinase IV, the O-GlcNAc-6-phosphate modification was found in the vicinity of two autophosphorylation sites required for full activation of the kinase suggesting a potential regulatory role for O-GlcNAc-6-phosphate. By re-analyzing mass spectrometric data from human embryonic and induced pluripotent stem cells, our study also identified Zinc finger protein 462 (ZNF462) as the first human O-GlcNAc-6-phosphate modified protein. Collectively, the data suggests that O-GlcNAc-6-phosphate is a general post-translation modification of mammalian proteins with a variety of possible cellular functions.Molecular & Cellular Proteomics 07/2012; 11(10):1063-9. · 7.25 Impact Factor
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ABSTRACT: Phosphorylation site assignment of high throughput tandem mass spectrometry (LC-MS/MS) data is one of the most common and critical aspects of phosphoproteomics. Correctly assigning phosphorylated residues helps us understand their biological significance. The design of common search algorithms (such as Sequest, Mascot etc.) do not incorporate site assignment; therefore additional algorithms are essential to assign phosphorylation sites for mass spectrometry data. The main contribution of this study is the design and implementation of a linear time and space dynamic programming strategy for phosphorylation site assignment referred to as PhosSA. The proposed algorithm uses summation of peak intensities associated with theoretical spectra as an objective function. Quality control of the assigned sites is achieved using a post-processing redundancy criteria that indicates the signal-to-noise ratio properties of the fragmented spectra. The quality assessment of the algorithm was determined using experimentally generated data sets using synthetic peptides for which phosphorylation sites were known. We report that PhosSA was able to achieve a high degree of accuracy and sensitivity with all the experimentally generated mass spectrometry data sets. The implemented algorithm is shown to be extremely fast and scalable with increasing number of spectra (we report up to 0.5 million spectra/hour on a moderate workstation). The algorithm is designed to accept results from both Sequest and Mascot search engines. An executable is freely available at http://helixweb.nih.gov/ESBL/PhosSA/ for academic research purposes.Proteome Science 11/2013; 11(Suppl 1):S14. · 2.42 Impact Factor
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ABSTRACT: The unsheathed flagellar filament of Shewanella oneidensis MR-1 is composed of two highly homologous flagellins, FlaA, and the major structural unit, FlaB. We identified a gene cluster, SO_3261-SO_3265 (now sfmABCDE), that is required for the formation of a fully functional filament and for motility. The predicted function of the corresponding gene products strongly indicated a role in flagellin modification. Accordingly, loss of sfmABCDE results in a significant mass shift of both FlaA and FlaB. Mass spectroscopy analysis and single residue substitutions identified five serine residues in both flagellins that are modified via O-linkage. Modeling of the flagellin structures strongly suggests that at least four of the modified residues are exposed to the filament's surface. However, none of the five serine residues solely is crucial for function and assembly. Structural analysis of the flagellin modification revealed that it likely contains a nonulosonic acid (274 Da) linked to each glycosylated serine. The putative nonulosonic acid is further substituted with a 236 Da moiety which can carry additional methyl groups (250 Da, 264 Da). In addition, at least 5 lysine residues in FlaB and one in FlaA were found to be methylated. Based on homology comparisons we suggest that smfABCDE is required for species-specific flagellin modification in S. oneidensis MR-1.PLoS ONE 01/2013; 8(9):e73444. · 3.53 Impact Factor