Discovery of protein acetylation patterns by deconvolution of peptide isomer mass spectra

Abstract

Protein post-translational modifications (PTMs) play important roles in the control of various biological processes including protein–protein interactions, epigenetics and cell cycle regulation. Mass spectrometry-based proteomics approaches enable comprehensive identification and quantitation of numerous types of PTMs. However, the analysis of PTMs is complicated by the presence of indistinguishable co-eluting isomeric peptides that result in composite spectra with overlapping features that prevent the identification of individual components. In this study, we present Iso-PeptidAce, a novel software tool that enables deconvolution of composite MS/MS spectra of isomeric peptides based on features associated with their characteristic fragment ion patterns. We benchmark Iso-PeptidAce using dilution series prepared from mixtures of known amounts of synthetic acetylated isomers. We also demonstrate its applicability to different biological problems such as the identification of site-specific acetylation patterns in histones bound to chromatin assembly factor-1 and profiling of histone acetylation in cells treated with different classes of HDAC inhibitors.

Identification of absolute geometries of cis and trans molecular isomers by Coulomb Explosion Imaging

“…molecules with the same chemical formula but different geometric structures, play an important role in many biological processes123456. For example, our vision requires a protein called rhodopsin, which inter-converts between different geometric structures (i.e.…”

Deconvolution in mass spectrometry based proteomics

“…Most of the above‐mentioned methods were intended to distinguish co‐eluting peptides with different sequences from MS/MS spectra. For example, an approach that modeled the product ion patterns into a network flow problem was used to deconvolute the product ion spectra of acetylated isomers, which have the same sequence and the same acetylated modification at different sites . Another program, Xim, was used to attempt to identify peptide isomers in a MS/MS spectrum through specified peptide modification rules and a score strategy, which computed a separation score to take the mixture of two isomers into account …”

“…For example, an approach that modeled the product ion patterns into a network flow problem was used to deconvolute the product ion spectra of acetylated isomers, which have the same sequence and the same acetylated modification at different sites. 65 Another program, Xim, was used to attempt to identify peptide isomers in a MS/MS spectrum through specified peptide modification rules and a score strategy, which computed a separation score to take the mixture of two isomers into account. 66 Apart from the co-eluting problems, other drawbacks of DDA included poor reproducibility and limited sensitivity in precursor ion detection that was influenced by background noise, as well as imprecise precursor ion peptide sampling proportion in the elution profile.…”

Epiproteomics: quantitative analysis of histone marks and codes by mass spectrometry

“…The number of equations formulated from ratios of MS2 measurement is fewer than the parameters needed to be solved for ratios of these isomers. Recent efforts used MS3 (refragmentation of an isolated MS2 fragment ion) [38 •• ] and unique fragment ion patterns associated with each isomer to deconvolute them [39 •• ]. (c) When two methyl groups are detected in MS2 fragment ions with multiple lysines, it could be a dimethylation on a single lysine or two monomethylation on two residues.…”

“…Two strategies have been reported recently to solve the long-standing problem of four ‘positional isomers’ of a diacetylated H4 (Figure 3b, [27]). The first one used an MS3 approach [38 •• ] and the second took advantage of the characteristic fragment ion pattern associated with each positional isomer [39 •• ]. This isobaric problem grows more complicated when methylation is involved.…”