Narrowband Modulation Two-Dimensional Mass Spectrometry and Label-Free Relative Quantification of Histone Peptides.

Matthias Halper,Maria A Van Agthoven,Marc-André Delsuc,Kathrin Breuker

doi:10.1021/acs.analchem.0c02843

Matthias Halper, Maria A Van Agthoven + Show 2 more

Open Access

https://doi.org/10.1021/acs.analchem.0c02843

Copy DOI

Abstract

Two-dimensional mass spectrometry (2D MS) on a Fourier transform ion cyclotron resonance (FT-ICR) mass analyzer allows for tandem mass spectrometry without requiring ion isolation. In the ICR cell, the precursor ion radii are modulated before fragmentation, which results in modulation of the abundance of their fragments. The resulting 2D mass spectrum enables a correlation between the precursor and fragment ions. In a standard broadband 2D MS, the range of precursor ion cyclotron frequencies is determined by the lowest mass-to-charge (m/z) ratio to be fragmented in the 2D MS experiment, which leads to precursor ion m/z ranges that are much wider than necessary, thereby limiting the resolving power for precursor ions and the accuracy of the correlation between the precursor and fragment ions. We present narrowband modulation 2D MS, which increases the precursor ion resolving power by reducing the precursor ion m/z range, with the aim of resolving the fragment ion patterns of overlapping isotopic distributions. In this proof-of-concept study, we compare broadband and narrowband modulation 2D mass spectra of an equimolar mixture of histone peptide isoforms. In narrowband modulation 2D MS, we were able to separate the fragment ion patterns of all 13C isotopes of the different histone peptide forms. We further demonstrate the potential of narrowband 2D MS for label-free quantification of peptides.

Highlights

Histones are 11−21 kDa proteins that make up the chief components of chromatin, the complex around which DNA is wound in the nucleus.[1]
This study shows that decreasing the sampling frequency along the t1 axis in 2D Mass spectrometry (MS) can substantially increase the resolving power in the vertical precursor ion m/z dimension for an unchanged acquisition time
The precursor−fragment correlation was high enough to enable the detailed examination of fragment isotopic distributions in the 2D mass spectrum

Summary

Introduction

Histones are 11−21 kDa proteins that make up the chief components of chromatin, the complex around which DNA is wound in the nucleus.[1]. Mass spectrometry (MS) is ideally suited to address this challenge as it can directly detect all mass-altering modifications and does not require laborious biochemical techniques for analysis. MS can provide mass values of both the biomolecules under study and their fragments from dissociation in tandem mass spectrometry (MS/MS) experiments, for which prefractionation by chromatography is typically required.[3,4] it is often challenging if not impossible to fractionate very similar compounds, for example, post-translationally modified proteins that differ only in their modification patterns. Standard MS/MS experiments, in which each ionized compound (precursor ion) in a sample is isolated before dissociation and detection of its fragment ions in the mass spectrometer, can be limited by overlapping isotopic distributions of different peptide or protein forms. Isolation and fragmentation of a single histone isoform,[5] as well as the label-free, direct localization and relative quantitation of histone[6−8] and ribonucleic acid,[8] modifications by MS/MS have been demonstrated, a more general approach that does not rely on precursor ion isolation would significantly advance all fields of PTM research

Methods

Results

Conclusion