Analytical utility of mass spectral binning in proteomic experiments by SPectral Immonium Ion Detection (SPIID).
Bottom Line: Although such ions offer tremendous analytical advantages, algorithms to decipher MS/MS spectra for the presence of diagnostic ions in an unbiased manner are currently lacking.To benchmark the software tool, we analyzed large higher-energy collisional activation dissociation datasets of samples containing phosphorylation, ubiquitylation, SUMOylation, formylation, and lysine acetylation.Using the developed software tool, we were able to identify known diagnostic ions by comparing histograms of modified and unmodified peptide spectra.
Affiliation: From the ‡Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Faculty of Health Sciences, DK-2200 Copenhagen, Denmark;Show MeSH
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Mentions: The graphical user interface of SPIID is shown in Fig. 4A. The input required for SPIID is high-resolution MS/MS peak lists following the standard Mascot generic format (MGF). In the MGF file, each MS/MS spectrum is listed as a pair of mass and intensity values delimited by “BEGIN IONS” and “END IONS” statements (59), and is commonly regarded the most used file format for storing MS/MS data (60). The MGF input files can be generated through standard proteomics software tools such as Mascot Distiller, MassMatrix (61), Raw2MSM (62), Pyteomics (63), or msconvert (ProteoWizard) (64). Notably, the apl peak lists generated by MaxQuant are also supported. First (i) the user loads the MGF file into the SPIID program by clicking “Add MGF file.” This can be followed by optional (ii) loading of a specificity file containing information regarding the desired grouping and the raw files and scan numbers. The next step (iii) is the optional choice of which spectra to use as background determinants in the analysis; typically these would be all non-modified spectra, but other options are made available as well. The last step (iv) before processing can begin is determining the bin size and start/stop m/z values for the analysis. Finally (v), pressing the “Process” button will initiate the SPIID analysis, and the output will be depicted as illustrated in Fig. 4B. The top pane of the output window portrays the binned histogram for all modified MS/MS spectra being analyzed, and the middle pane portrays a similar histogram derived from all unmodified MS/MS spectra. The bottom pane of the output window depicts the final normalized diagnostic ion histogram, generated by subtraction of the two upper histograms (modified histogram minus unmodified histogram). The entire right side of the output window allows for changing the visualized output style according to various parameters such as font size, color, histogram annotation, depicted m/z range, etc. This ensures flexibility for the end user and allows for visual optimization of data output. The final diagnostic ion histogram can be exported to several file formats.
Affiliation: From the ‡Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Faculty of Health Sciences, DK-2200 Copenhagen, Denmark;