Natural isotope correction improves analysis of protein modification dynamics

Abstract

Stable isotope labelling in combination with high-resolution mass spectrometry approaches are increasingly used to analyze both metabolite and protein modification dynamics. To enable correct estimation of the resulting dynamics, it is critical to correct the measured values for naturally occurring stable isotopes, a process commonly called isotopologue correction or deconvolution. While the importance of isotopologue correction is well recognized in metabolomics, it has received far less attention in proteomics approaches. Although several tools exist that enable isotopologue correction of mass spectrometry data, the majority is tailored for the analysis of low molecular weight metabolites. We here present PICor which has been developed for isotopologue correction of complex isotope labelling experiments in proteomics or metabolomics and demonstrate the importance of appropriate correction for accurate determination of protein modifications dynamics, using histone acetylation as an example.

Combined Metabolic and Chemical (CoMetChem) Labeling Using Stable Isotopes—a Strategy to Reveal Site-Specific Histone Acetylation and Deacetylation Rates by LC–MS

“…We therefore included an isotope correction in the data analysis to ensure accurate quantification. The Python-based isotope correction pipeline PICor used a skewed matrix algorithm, which corrects each different isotopologue species with its own set of theoretical correction factors . A detailed tutorial on bioinformatics workflow and data processing can be found in the Supporting Information.…”

“…For natural isotope abundance correction, the python package PICor was used, which uses a theoretical correction approach based on statistical distributions of each isotopologue. The detailed approach is described on bioRxiv, and the source code is available under . To determine site-specific acetylation levels for the MS1 isobaric H3­(18-26) isotopologue species and single-acetylated H4­(4-17) isotopologue species, the relative abundances of site-specific acetyllysine-containing fragment ions were quantified using Pipelines and Systems for Threshold-Avoiding Quantification of LC–MS/MS data (PASTAQ) .…”

“…The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.1c01359. MS1 information of H3 (18)(19)(20)(21)(22)(23)(24)(25)(26)…”

“…For natural isotope abundance correction, the python package PICor was used, which uses a theoretical correction approach based on statistical distributions of each isotopologue. The detailed approach is described on bioRxiv, 25 and the source c o d e i s a v a i l a b l e u n d e r h t t p s : / / g i t h u b . c o m / MolecularBioinformatics/PICor.…”

“…Comparison of the derivatization efficiency of intact histones, identification and quantification of H3[18][19][20][21][22][23][24][25][26] positional isomers, site-specific abundance of the singleand double-acetylated H3(18-26) peptide species, initial flux analysis to determine reaction rates of acetylation and deacetylation, effect of the histone deacetylase inhibitors MS-275 and SAHA on the H4(4-17) acetylation level, chemical structure of the HDAC inhibitors and the workflow for the metabolic labeling, site-specific label incorporation for K18ac and K23ac of the single-and double-acetylated H3(18-26) species, flowchart showing the different steps of the CoMet-Chem data analysis pipeline, MS1 spectrum of the H3(18-26) isotopologues, extracted ion chromatograms (EICs) of the H3(18-26) isotopologue species, abundances of H3(18-26) isotopologues before and after PICor correction, graphical user interface (GUI) of PASTAQ, gaussian fitting of detected y7-ion fragments, site-specific label incorporation and label loss of the single-acetylated H3(18)(19)(20)(21)(22)(23)(24)(25)(26), linear fit over the abundances of the single-12C acetylated species, calculated half-lives of acetylated H3(18-26) species, calculated reaction rates of acetylation and deacetylation of the H3(18)(19)(20)(21)(22)(23)(24)(25)(26) species, calculated reaction rates of acetylation and deacetylation of the single-acetylated H4(4-17) species, intensities of the H3(18-26) isotopologues, input for PICor correction, sample table for MS2 fragment ion quantification, fragment table fragment ion quantification, and fragment ion quantification results (PDF)…”