Analytical Utility of Mass Spectral Binning in Proteomic Experiments by SPectral Immonium Ion Detection (SPIID)

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.

Global mapping of post-translational modifications on histone H3 variants in mouse testes

“…5 ). Formylation specifically forms immonium ion at m/z 112 compared with di-methylation, as reported in a previous study [32] , [33] . Formylation of immonium ion at m/z 112.076 was also observed in our results ( Fig.…”

ProteomeTools: Systematic Characterization of 21 Post-translational Protein Modifications by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) Using Synthetic Peptides

“…presented a tool for spectral binning to identify masses exclusive for a given PTM compared to unmodified peptides (37). Following up on and extending this idea, we implemented an intensity weighted m/z aggregation strategy for fragment ions originating from thousands of spectra and for all the 21 modifications and fragmentation modes used in our study.…”

Enzymology and significance of protein histidine methylation

“…It was recently reported that Hme-bearing peptides can give rise to a specific immonium ion in tandem mass spectra upon fragmentation ( 12 ). The Hme immonium ion has a mass of 124.087 atomic mass units, a region with low background signal in mass spectra ( 92 ). Inspection of mass spectra for this immonium ion can corroborate Hme events and enable Hme bearing peptides to be distinguished from those with SNPs or other monomethylation events.…”