Comparison of data‐acquisition methods for the identification and quantification of histone post‐translational modifications on a Q Exactive HF hybrid quadrupole Orbitrap mass spectrometer

Abstract

Rationale Histone post‐translational modifications (PTMs) play key roles in regulating eukaryotic gene expression. Mass spectrometry (MS) has emerged as a powerful method to characterize and quantify histone PTMs as it allows unbiased identification and quantification of multiple histone PTMs including combinations of the modifications present. Methods In this study we compared a range of data‐acquisition methods for the identification and quantification of the histone PTMs using a Q Exactive HF Orbitrap. We compared three different data‐dependent analysis (DDA) methods with MS2 resolutions of 120K, 60K, 30K. We also compared a range of data‐independent analysis (DIA) methods using MS2 isolation windows of 20 m/z and DIAvw to identify and quantify histone PTMs in Chinese hamster ovary (CHO) cells. Results The increased number of MS2 scans afforded by the lower resolution methods resulted in a higher number of queries, peptide sequence matches (PSMs) and a higher number of peptide proteoforms identified with a Mascot Ion score greater than 46. No difference in the proportion of peptide proteoforms with Delta scores >17 was observed. Lower coefficients of variation (CVs) were obtained in the DIA MS1 60 K MS2 30 K 20 m/z isolation windows compared with the other data‐acquisition methods. Conclusions We observed that DIA which offers advantages in flexibility and identification of isobaric peptide proteoforms performs as well as DDA in the analysis of histone PTMs. We were able to identify 71 modified histone peptides for histone H3 and H4 and quantified 64 across each of the different acquisition methods.

Rapid qualitative profiling of metabolites present in Fusarium solani, a rhizospheric fungus derived from Senna spectabilis, using GC/MS and UPLC-QTOF/MSE techniques assisted by UNIFI information system

“…In this sense, the DIA analyzes overcome some limitations of the analyzes in DDA. 47 Some authors cite the difficulty of DIA analysis for some types of samples, such as lipids, which can lose the connection between the precursor ion and the product ions, 48 which does not happen in this work. Although the MS E analysis method is widely used in proteomics, 49 it has also been broadly used in metabolomics.…”

Pneumolysin Is Responsible for Differential Gene Expression and Modifications in the Epigenetic Landscape of Primary Monocyte Derived Macrophages

“…The peptides were re-suspended in 0.1%TFA and 3% ACN and loaded into and run on Ultimate 3000 RSLC nano flow liquid chromatography system with a PepMap300 C18 trapping column (Thermo Fisher), coupled to Q-Exactive HF Orbitrap mass spectrometer (Thermo Fisher). Peptides were eluted onto a 50 cm x 75 μm Easy-spray PepMap C18 column with a flow rate of 300 nL/min as previously described ( 35 ). Peptides were eluted using a gradient of 3% to 35% solvent B over 75 min.…”

Proteoforms: General Concepts and Methodological Process for Identification

“…Tandem MS can confirm the protein identification based on the daughter ions and characteristics of the obtained peptide map and primary structure, which thereafter provide exact localization of post-translational or other modification sites. Data-independent acquisition (DIA) methods have been alternatively used to analyze proteoforms particularly suited to the study of PTMs [72]. DIA focuses on the identification and quantitation of fragment ions that are generated from multiple peptides contained in the same selection window of several to tens of m/z, that is, the fragmentation spectra of all the peptides are acquired in each cycle time without any preselection of the precursor ions [73].…”