|Simone Sidoli Ph.D. works in the laboratory directed by Prof. Garcia (University of Pennsylvania), where we focus on the analysis of histone PTMs using mass spectrometry. Since several histone peptides have isobaric forms, a central part of our work consists in accurately discriminating their abundance using the profiles of selected fragment ions. Read More |
We frequently use Skyline to retrieve fragment ion profiles, and now we developed templates to analyze more than 200 known histone modifications and protein phosphorylation in general. We believe we are taking advantage of a feature of Skyline that is currently unexploited by proteomics labs in general. We would like to show that critical insights are missed when isobaric forms are not considered.
DIA for Differential Quantification of Isobaric Phosphopeptides and Other Protein Post-translational Modifications
State-of-the-art quantification in proteomics is performed by extracting the precursor ion chromatogram of identified peptides. When using data independent acquisition (DIA) the profiles of the fragment ions can also be used to increase quantification confidence, or sometimes in alternative to the precursor ion signal. We recently demonstrated that in both phosphoproteomics (PubMedID: 27301801) and histone peptide analysis (PubMedID: 26505526) many modified peptides are erroneously quantified using both approaches, because they have isobaric forms that cannot be discriminated when co-eluting by liquid chromatography. These peptides have the same mass, but modifications on different residues. We thus developed templates that use DIA data and the Skyline (v3.6) output to assign the correct intensity on co-eluting isobaric modified peptides. These templates select unique fragment ions to determine the relative ratio of such peptides. This approach is imperative for samples where isobaric forms are common, i.e. histone peptides, for proper biological interpretation.