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

Abstract

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.

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

“…Since then, the development of 2D MS and the urQRd algorithm have enabled applications in the analysis of small molecules, proteomics (both top-down and bottom-up), and polymers (Floris et al 2016 , 2017 , 2018c ; Simon et al 2016 ; van Agthoven et al 2015 ; van Agthoven et al 2016 ). 2D MS has also been applied for MS 3 experiments on proteins without ion isolation (Floris et al 2018a , b ). Because of the multiplexing of the data inherent to Fourier analysis, 2D MS has capabilities that other DDA and DIA techniques do not have in terms of correlation between precursor and fragment ions, especially for overlapping isotopic distributions (van Agthoven et al 2016 ).…”

Phase relationships in two-dimensional mass spectrometry

“…As Eqs. (18) and (19) show, c 2 , c 1 ′, and c 0 do not depend on t 1 , but the phase function of precursor ions is dependent on t 1 and precursor ion packets have a different phase function for each transient that is recorded in the 2D MS experiment. This phase function only applies if ions are not de-excited back to the ICR cell after the second pulse.…”

“…The comparison between Eqs. (18), (19), and (20) shows that precursor and fragment ions have different phase values. In addition, sometimes the same ion m/z can be a precursor present at the start of the pulse sequence and a fragment generated from another precursor during the fragmentation period.…”

“…Denoising algorithms have been proposed in order to decrease the amount of scintillation noise in 2D mass spectra and the pulse sequence was optimized [15][16][17]. 2D MS has been applied to small molecules and bottom-up and top-down proteomics, as well as polymer analysis [18][19][20][21][22][23][24][25]. Automated peak-assignment algorithms are being developed for routine analysis [26].…”

Proteomics and proteoforms: Bottom-up or top-down, how to use high-resolution mass spectrometry to reach the Grail

“…The main recent developments for peptide analysis concern two-dimensional FTMS approaches, introduced on glycosylated peptides [53]. The first analyses in bottom-up data-independent acquisition (DIA) mode were performed on cytochrome C [54] and subsequently on calmodulin and ubiquitin [55,56]. This very promising approach exploits the possibility of correlating the precursor ion and its fragments via the ion of interest's cyclotron frequency modulation by avoiding any selection step.…”