Top-Down Proteoform Analysis by 2D MS with Quadrupolar Detection.

Two-dimensional mass spectrometry (2D MS) is a multiplexed tandem mass spectrometry method that does not rely on ion isolation to correlate the precursor and fragment ions. On a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS), 2D MS instead uses the modulation of precursor ion radii inside the ICR cell before fragmentation and yields 2D mass spectra that show the fragmentation patterns of all the analytes. In this study, we perform 2D MS for the first time with quadrupolar detection in a dynamically harmonized ICR cell. We discuss the advantages of quadrupolar detection in 2D MS and how we adapted existing data processing techniques for accurate frequency-to-mass conversion. We apply 2D MS with quadrupolar detection to the top-down analysis of covalently labeled ubiquitin with ECD fragmentation, and we develop a workflow for label-free relative quantification of biomolecule isoforms in 2D MS.

Ultra-Accurate Correlation between Precursor and Fragment Ions in Two-Dimensional Mass Spectrometry: Acetylated vs Trimethylated Histone Peptides.

Two-dimensional mass spectrometry (2D MS) is a method for tandem mass spectrometry in which precursor and fragment ions are correlated by manipulating ion radii rather than by ion isolation. A 2D mass spectrum contains the fragmentation patterns of all analytes in a sample, acquired in parallel. We report ultrahigh-resolution narrowband 2D mass spectra of a mixture of two histone peptides with the same sequence, one of which carries an acetylation and the other a trimethylation (m/z 0.006 difference). We reduced the distance between data points in the precursor ion dimension and compared the accuracy of the precursor-fragment correlation with the resolving power. We manage to perform label-free quantification on the histone peptide mixture and show that precursor and fragment ions can be accurately correlated even though the precursor ions are not resolved. Finally, we show that increasing the resolution of a 2D mass spectrum in the precursor ion dimension too far can lead to a decline in the signal-to-noise ratio.

Phase Correction for Absorption Mode Two-Dimensional Mass Spectrometry.

Two-dimensional mass spectrometry (2D MS) is a tandem mass spectrometry method that relies on manipulating ion motions to correlate precursor and fragment ion signals. 2D mass spectra are obtained by performing a Fourier transform in both the precursor ion mass-to-charge ratio (m/z) dimension and the fragment ion m/z dimension. The phase of the ion signals evolves linearly in the precursor m/z dimension and quadratically in the fragment m/z dimension. This study demonstrates that phase-corrected absorption mode 2D mass spectrometry improves signal-to-noise ratios by a factor of 2 and resolving power by a factor of 2 in each dimension compared to magnitude mode. Furthermore, phase correction leads to an easier differentiation between ion signals and artefacts, and therefore easier data interpretation.

Application of Optimal Control Theory to Fourier Transform Ion Cyclotron Resonance.

We study the application of Optimal Control Theory to Ion Cyclotron Resonance. We test the validity and the efficiency of this approach for the robust excitation of an ensemble of ions with a wide range of cyclotron frequencies. Optimal analytical solutions are derived in the case without any pulse constraint. A gradient-based numerical optimization algorithm is proposed to take into account limitation in the control intensity. The efficiency of optimal pulses is investigated as a function of control time, maximum amplitude and range of excited frequencies. A comparison with adiabatic and SWIFT pulses is done. On the basis of recent results in Nuclear Magnetic Resonance, this study highlights the potential usefulness of optimal control in Ion Cyclotron Resonance.

Self-organization Properties of a GPCR-Binding Peptide with a Fluorinated Tail Studied by Fluorine NMR Spectroscopy.

Conjugation of the bioactive apelin-17 peptide with a fluorocarbon chain results in self-organization of the peptide into micelles. Fluorine NMR spectroscopy studies show that the fluoropeptide's micelles are monodisperse, while proton NMR indicates that the peptide moiety remains largely disordered despite micellization. A very fast exchange rate is measured between the free and micellar states of the peptide which enables the number of molecules present in the micelle to be estimated as 200, in agreement with values found by dynamic light scattering measurements.

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

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.

Phase relationships in two-dimensional mass spectrometry.

Two-dimensional mass spectrometry (2D MS) is a data-independent tandem mass spectrometry technique in which precursor and fragment ion species can be correlated without the need for prior ion isolation. The behavior of phase in 2D Fourier transform mass spectrometry is investigated with respect to the calculation of phase-corrected absorption-mode 2D mass spectra. 2D MS datasets have a phase that is defined differently in each dimension. In both dimensions, the phase behavior of precursor and fragment ions is found to be different. The dependence of the phase for both precursor and fragment ion signals on various parameters (e.g., modulation frequency, shape of the fragmentation zone) is discussed. Experimental data confirms the theoretical calculations of the phase in each dimension. Understanding the phase relationships in a 2D mass spectrum is beneficial to the development of possible algorithms for phase correction, which may improve both the signal-to-noise ratio and the resolving power of peaks in 2D mass spectra.

Targeting bioactive compounds in natural extracts - Development of a comprehensive workflow combining chemical and biological data.

In natural product drug discovery, several strategies have emerged to highlight specifically bioactive compound(s) within complex mixtures (fractions or crude extracts) using metabolomics tools. In this area, a great deal of interest has raised among the scientific community on strategies to link chemical profiles and associated biological data, leading to the new field called "biochemometrics". This article falls into this emerging research by proposing a complete workflow, which was divided into three major steps. The first one consists in the fractionation of the same extract using four different chromatographic stationary phases and appropriated elution conditions to obtain five fractions for each column. The second step corresponds to the acquisition of chemical profiles using HPLC-HRMS analysis, and the biological evaluation of each fraction. The last step evaluates the links between the relative abundances of molecules present in fractions (peak area) and the global bioactivity level observed for each fraction. To this purpose, an original bioinformatics script (encoded with R Studio software) using the combination of four statistical models (Spearman, F-PCA, PLS, PLS-DA) was here developed leading to the generation of a "Super list" of potential bioactive compounds together with a predictive score. This strategy was validated by its application on a marine-derived Penicillium chrysogenum extract exhibiting antiproliferative activity on breast cancer cells (MCF-7 cells). After the three steps of the workflow, one main compound was highlighted as responsible for the bioactivity and identified as ergosterol. Its antiproliferative activity was confirmed with an IC50 of 0.10 µM on MCF-7 cells. The script efficiency was further demonstrated by comparing the results obtained with a different recently described approach based on NMR profiling and by virtually modifying the data to evaluate the computational tool behaviour. This approach represents a new and efficient tool to tackle some of the bottlenecks in natural product drug discovery programs.

Automatised pharmacophoric deconvolution of plant extracts - application to Cinchona bark crude extract.

We present a development of the "Plasmodesma" dereplication method [Margueritte et al., Magn. Reson. Chem., 2018, 56, 469]. This method is based on the automatic acquisition of a standard set of NMR experiments from a medium sized set of samples differing by their bioactivity. From this raw data, an analysis pipeline is run and the data is analysed by leveraging machine learning approaches in order to extract the spectral fingerprints of the active compounds. The optimal conditions for the analysis are determined and tested on two different systems, a synthetic sample where a single active molecule is to be isolated and characterized, and a complex bioactive matrix with synergetic interactions between the components. The method allows the identification of the active compounds and performs a pharmacophoric deconvolution. The program is freely available on the Internet, with an interactive visualisation of the statistical analysis, at https://plasmodesma.igbmc.science.

Two-dimensional mass spectrometry: new perspectives for tandem mass spectrometry.

Fourier transform ion cyclotron resonance mass analysers (FT-ICR MS) can offer the highest resolutions and mass accuracies in mass spectrometry. Mass spectra acquired in an FT-ICR MS can yield accurate elemental compositions of all compounds in a complex sample. Fragmentation caused by ion-neutral, ion-electron, or ion-photon interactions leads to more detailed structural information on compounds. The most often used method to correlate compounds and their fragment ions is to isolate the precursor ions from the sample before fragmentation. Two-dimensional mass spectrometry (2D MS) offers a method to correlate precursor and fragment ions without requiring precursor isolation. 2D MS therefore enables easy access to the fragmentation patterns of all compounds from complex samples. In this article, the principles of FT-ICR MS are reviewed and the 2D MS experiment is explained. Data processing for 2D MS is detailed, and the interpretation of 2D mass spectra is described.

Application of Tandem Two-Dimensional Mass Spectrometry for Top-Down Deep Sequencing of Calmodulin.

Two-dimensional mass spectrometry (2DMS) involves simultaneous acquisition of the fragmentation patterns of all the analytes in a mixture by correlating their precursor and fragment ions by modulating precursor ions systematically through a fragmentation zone. Tandem two-dimensional mass spectrometry (MS/2DMS) unites the ultra-high accuracy of Fourier transform ion cyclotron resonance (FT-ICR) MS/MS and the simultaneous data-independent fragmentation of 2DMS to achieve extensive inter-residue fragmentation of entire proteins. 2DMS was recently developed for top-down proteomics (TDP), and applied to the analysis of calmodulin (CaM), reporting a cleavage coverage of about ~23% using infrared multiphoton dissociation (IRMPD) as fragmentation technique. The goal of this work is to expand the utility of top-down protein analysis using MS/2DMS in order to extend the cleavage coverage in top-down proteomics further into the interior regions of the protein. In this case, using MS/2DMS, the cleavage coverage of CaM increased from ~23% to ~42%. Graphical Abstract Two-dimensional mass spectrometry, when applied to primary fragment ions from the source, allows deep-sequencing of the protein calmodulin.

Top-Down Deep Sequencing of Ubiquitin Using Two-Dimensional Mass Spectrometry.

Two-dimensional mass spectrometry (2DMS) allows data independent fragmentation of all ions in a sample and correlation of fragment ions to their precursors without isolation prior to fragmentation. Developments in computer capabilities and implementations in Fourier transform ion cyclotron resonance (FTICR) MS over the past decade have allowed the technique to become a useful analytical tool for bottom-up proteomics (BUP) and, more recently, in top-down protein analysis (TDP). In this work, a new method of TDP is developed using 2D FTICR MS, called MS/2D FTICR MS or MS/2DMS. In MS/2DMS, an entire protein is initially fragmented in a hexapole collision cell, e.g., with collisionally activated dissociation (CAD). The primary fragments are then sent to the ICR cell, where 2DMS is performed with infrared multiphoton dissociation (IRMPD) or electron-capture dissociation (ECD). The resulting 2D mass spectra retain information equivalent to a set of TDP MS3 experiments on the selected protein. Up to n - 1 fragmentation steps can be added to the process, as long as an ion of interest can be unambiguously fragmented before the ICR cell, leading to an MS n/2DMS experiment whose output is a 2D mass spectrum retaining information equivalent to MS n. MS/2DMS and MS/MS/2DMS are used in this work for the structural analysis of ubiquitin (Ubi), noting several unique features which aid fragment identification. The use of CAD-MS/IRMPD-2DMS, CAD-MS/ECD-2DMS, and MS2/2DMS using, respectively, in-source dissociation (ISD), CAD, and ECD-2DMS led to 97% cleavage coverage for Ubi.

Can Two-Dimensional IR-ECD Mass Spectrometry Improve Peptide de Novo Sequencing?

Two-dimensional mass spectrometry (2D MS) correlates precursor and fragment ions without ion isolation in a Fourier transform ion cyclotron resonance mass spectrometer (FTICR MS) for tandem mass spectrometry. Infrared activated electron capture dissociation (IR-ECD), using a hollow cathode configuration, generally yields more information for peptide sequencing in tandem mass spectrometry than ECD (electron capture dissociation) alone. The effects of the fragmentation zone on the 2D mass spectrum are investigated as well as the structural information that can be derived from it. The enhanced structural information gathered from the 2D mass spectrum is discussed in terms of how de novo peptide sequencing can be performed with increased confidence. 2D IR-ECD MS is shown to sequence peptides, to distinguish between leucine and isoleucine residues through the production of w ions as well as between C-terminal ( b/ c) and N-terminal ( y/ z) fragments through the use of higher harmonics, and to assign and locate peptide modifications.

Bottom-Up Two-Dimensional Electron-Capture Dissociation Mass Spectrometry of Calmodulin.

Two-dimensional mass spectrometry (2D MS) is a tandem mass spectrometry technique that allows data-independent fragmentation of all precursors in a mixture without previous isolation, through modulation of the ion cyclotron frequency in the ICR-cell prior to fragmentation. Its power as an analytical technique has been proven particularly for proteomics. Recently, a comparison study between 1D and 2D MS has been performed using infrared multiphoton dissociation (IRMPD) on calmodulin (CaM), highlighting the capabilities of the technique in both top-down (TDP) and bottom-up proteomics (BUP). The goal of this work is to expand this study on CaM using electron-capture dissociation (ECD) 2D MS as a single complementary BUP experiment in order to enhance the cleavage coverage of the protein under analysis. By adding the results of the BUP 2D ECD MS to the 2D IRMPD MS analysis of CaM, the total cleavage coverage increased from ~40% to ~68%. Graphical abstract ᅟ.

Automatic differential analysis of NMR experiments in complex samples.

Liquid state nuclear magnetic resonance (NMR) is a powerful tool for the analysis of complex mixtures of unknown molecules. This capacity has been used in many analytical approaches: metabolomics, identification of active compounds in natural extracts, and characterization of species, and such studies require the acquisition of many diverse NMR measurements on series of samples. Although acquisition can easily be performed automatically, the number of NMR experiments involved in these studies increases very rapidly, and this data avalanche requires to resort to automatic processing and analysis. We present here a program that allows the autonomous, unsupervised processing of a large corpus of 1D, 2D, and diffusion-ordered spectroscopy experiments from a series of samples acquired in different conditions. The program provides all the signal processing steps, as well as peak-picking and bucketing of 1D and 2D spectra, the program and its components are fully available. In an experiment mimicking the search of a bioactive species in a natural extract, we use it for the automatic detection of small amounts of artemisinin added to a series of plant extracts and for the generation of the spectral fingerprint of this molecule. This program called Plasmodesma is a novel tool that should be useful to decipher complex mixtures, particularly in the discovery of biologically active natural products from plants extracts but can also in drug discovery or metabolomics studies.

Nonuniform Sampling Acquisition of Two-Dimensional Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Increased Mass Resolution of Tandem Mass Spectrometry Precursor Ions.

Obtaining the full MS/MS map for fragments and precursors of complex mixtures without hyphenation with chromatographic separation by a data-independent acquisition is a challenge in mass spectrometry which is solved by two-dimensional (2D) Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). Until now 2D FTICR MS afforded only a moderate resolution for precursor ion since this resolution is limited by the number of evolution interval steps to which the number of scans, the acquisition time, and the sample consumption are proportional. An overnight acquisition is already required to reach a quadrupole mass filter-like unit mass resolution. Here, we report that 2D FTICR MS using nonuniform sampling (NUS) obtained by randomly skipping points in the first dimension corresponding to the precursor selection gives access, after data processing, to the same structural information contained in a complex mixture. The resolution increases roughly as the inverse of the NUS ratio, up to 26 times at NUS 1/32, leading to an acquisition time reduced in the same ratio compared to a classical acquisition at the same resolution. As an example, the analysis of a natural oil is presented.

Polymer Analysis in the Second Dimension: Preliminary Studies for the Characterization of Polymers with 2D MS.

Two-dimensional Fourier transform ion cyclotron resonance mass spectrometry (2D FTICR MS or 2D MS) allows direct correlation between precursor and fragment ions without isolation prior to fragmentation. The method has been optimized for the analysis of complex mixtures and used so far for the analysis of small molecules and peptides obtained by tryptic digestion of proteins and entire proteins. In this work, a 2D MS method is developed to characterize complex mixtures of polymers using infrared multiphoton decay (IRMPD) and electron capture dissociation (ECD) as fragmentation techniques, and D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), Polysorbate 80, and poly(methyl methacrylate) (PMMA) as analytes. The use of 2D MS allowed generation of fragment m/z values for all the compounds in the mixture at once and allowed tandem mass spectrometry of species very close in m/z that would have been difficult to isolate with a quadrupole for standard MS/MS. Furthermore, the use of unique features of 2D MS such as the extraction of neutral-loss lines allowed the successful assignment of peaks from low abundant species that would have been more difficult with standard MS/MS. For all the samples, the amount of information obtained with 2D MS was comparable with what obtained with multiple 1D MS/MS experiments targeted on each individual component within each mixture but required a single experiment of about 20-40 min.