Abstract
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 Two-dimensional mass spectrometry, when applied to primary fragment ions from the source, allows deep-sequencing of the protein calmodulin.
Highlights
The complexity of protein forms has expanded the need for proteomics [1, 2], and technologies such as mass spectrometry (MS) offer a leading platform for characterization of such macromolecules [3]
The autocorrelation line is extracted from the CAD-MS/ infrared multiphoton dissociation (IRMPD)-2DMS mass spectrum and reported in Figure 2.b, showing a large number of b/y ions, neutral losses, and internal fragments: typical ion fragments generated by techniques such as CAD
MS/2DMS is a promising tool for deep sequencing of proteins, providing a new fragmentation tool for top-down proteomics
Summary
The complexity of protein forms has expanded the need for proteomics [1, 2], and technologies such as mass spectrometry (MS) offer a leading platform for characterization of such macromolecules [3]. Studying proteins through MS in their entirety (Top-Down, TDP), rather than from smaller peptides obtained through their enzymatic digestion (Bottom-Up Proteomics, BUP), offers the highest amount of structural information, but constitutes a more challenging experiment. F. Floris et al.: Application of MS/2DMS to Top-down Deep Sequencing of CaM mass spectra such as urQRd [17], and computational [18] and tuning optimizations [19, 20]. The technique has been expanded for use in linear ion traps [26] and applications in polymer analysis [27]
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