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: In the next step each modified spectrum is further subdivided into specific PTM categories (e.g. lysine-acetylation-containing spectra are separated into a separate subcategory), and for each PTM subcategory all MS/MS spectra are mass binned across the preset m/z range, thereby creating fragment-specific histograms for each analyzed category (as shown in Fig. 2C). The binning histogram is generated arbitrarily from a starting m/z mass value, and this is followed by the generation of discrete mass bins, which can be defined by the user. The size of the mass bin can be varied accordingly but is preset to 40 ppm because of the high-accuracy fragment ions generated by HCD. The default value works well in our hands but can easily be adjusted in the SPIID software interface. Notably, the bin width is dependent on the mass accuracy of the instrumentation employed and can be affected by instrument-dependent factors such as calibration status and resolution settings.
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;