Abstract. Two-dimensional Fourier transform ion cyclotron resonance mass spectrometry (2D FT-ICR MS) allows data-independent fragmentation of all ions in a sample and correlation of fragment ions to their precursors through the modulation of precursor ion cyclotron radii prior to fragmentation. Previous results show that implementation of 2D FT-ICR MS with infrared multi-photon dissociation (IRMPD) and electron capture dissociation (ECD) has turned this method into a useful analytical tool. In this work, IRMPD tandem mass spectrometry of calmodulin (CaM) has been performed both in one-dimensional and two-dimensional FT-ICR MS using a top-down and bottom-up approach. 2D IRMPD FT-ICR MS is used to achieve extensive inter-residue bond cleavage and assignment for CaM, using its unique features for fragment identification in a less time-and sample-consuming experiment than doing the same thing using sequential MS/MS experiments.

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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.