Abstract
Unambiguous identification of tandem mass spectra is a cornerstone in mass-spectrometry-based proteomics. As the study of post-translational modifications (PTMs) by means of shotgun proteomics progresses in depth and coverage, the ability to correctly identify PTM-bearing peptides is essential, increasing the demand for advanced data interpretation. Several PTMs are known to generate unique fragment ions during tandem mass spectrometry, the so-called diagnostic ions, which unequivocally identify a given mass spectrum as related to a specific PTM. 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. Here, we present a systematic spectral-pattern-based approach for the discovery of diagnostic ions and new fragmentation mechanisms in shotgun proteomics datasets. The developed software tool is designed to analyze large sets of high-resolution peptide fragmentation spectra independent of the fragmentation method, instrument type, or protease employed. 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. Because the investigated tandem mass spectra data were acquired with high mass accuracy, unambiguous interpretation and determination of the chemical composition for the majority of detected fragment ions was feasible. Collectively we present a freely available software tool that allows for comprehensive and automatic analysis of analogous product ions in tandem mass spectra and systematic mapping of fragmentation mechanisms related to common amino acids.
Highlights
Automatic analysis of analogous product ions in tandem mass spectra and systematic mapping of fragmentation mechanisms related to common amino acids
The complex peptide mixture is separated via liquid chromatography (LC) directly coupled to mass spectrometry (MS), and the eluting peptide ions are electrosprayed into the vacuum of the mass spectrometer, where a peptide mass spectrum is recorded (2)
Description of Algorithm—For unbiased mapping of commonly occurring fragment ions, we developed an algorithm referred to as SPectral Immonium Ion Detection (SPIID) based upon binning of tandem mass spectra
Summary
Analytical Utility of Mass Spectral Binning in Proteomic Experiments by SPectral Immonium Ion Detection (SPIID)*□S. Several PTMs are known to generate unique fragment ions during tandem mass spectrometry, the socalled diagnostic ions, which unequivocally identify a given mass spectrum as related to a specific PTM. Low-energy dissociation conditions as observed in ion trap CID mainly generate fragment ions containing sequence-specific amino acid information about the investigated peptides (8). This occurs because the energy deposited during this fragmentation method primarily facilitates the fragmentation. Multiple bonds can thereby be fragmented, giving rise to internal sequence ions, which in combination with regular b- and y-type cleavage produce specific amino-immonium ions (11) These immonium ions appear in the very low m/z range of the MS/MS spectrum, and for the majority of naturally occurring amino acids such immonium ions are unique for that particular residue (12, 13). We demonstrate that mass spectral binning can be employed for automated mapping of composition-specific neutral losses from largescale proteomic experiments
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