Multimodal Tandem Mass Spectrometry Techniques for the Analysis of Phosphopeptides.

Collisionally activated dissociation (CAD), infrared multiphoton dissociation (IRMPD), ultraviolet photodissociation (UVPD), electron capture dissociation and electron detachment dissociation (EDD) experiments were conducted on a set of phosphopeptides, in a Fourier transform ion cyclotron resonance mass spectrometer. The fragmentation patterns were compared and varied according to the fragmentation mechanisms and the composition of the peptides. CAD and IRMPD produced similar fragmentation profiles of the phosphopeptides, while UVPD produced a large number of complementary fragments. Electron-based dissociation techniques displayed lower fragmentation efficiencies, despite retaining the labile phosphate group, and drastically different fragmentation profiles. EDD produced complex spectra whose interpretation proved challenging.

Characterization Across a Dispersity: Polymer Mass Spectrometry in the Second Dimension.

Due to the natural dispersity that is present in synthetic polymers, an added complexity is always present in the analysis of polymeric species. Tandem mass spectrometry analysis requires the isolation of individual precursors before a fragmentation event to allow the unambiguous characterization of these species and is not viable at certain levels of complexity due to achievable isolation widths. Two-dimensional mass spectrometry (2DMS) fragments ions and correlates fragments with their corresponding precursors without the need for isolation. In this study, 2DMS electron capture dissociation (ECD) fragmentation of a polyoxazoline and polyacrylamide species was carried out, resulting in the analysis of byproducts and individual polymer species without the use of chromatographic techniques. This study shows that 2DMS ECD is a powerful tool for the analysis of polyacrylamide and polyoxazoline species and offers a new dimension in the characterization of polymers.

Two-Dimensional Mass Spectrometry Analysis of IgG1 Antibodies.

Two-dimensional mass spectrometry (2DMS) is a new, and theoretically ideal, data-independent analysis tool, which allows the characterization of a complex mixture and was used in the bottom-up analysis of IgG1 for the identification of post-translational modifications. The new peak picking algorithm allows the distinction between chimeric peaks in proteomics. In this application, the processing of 2DMS data correlates fragments to their corresponding precursors, with fragments from precursors which are <0.1 m/z at m/z 840 easily resolved, without the need for quadrupole or chromatographic separation.

Facile Determination of Phosphorylation Sites in Peptides Using Two-Dimensional Mass Spectrometry.

Detection and characterization of phosphopeptides by infrared multiphoton dissociation two-dimensional mass spectrometry (IRMPD 2DMS) is shown to be particularly effective. A mixture of phosphopeptides was analyzed by 2DMS without any prior separation. 2DMS enables the data independent analysis of the mixture and the correlation of the fragments to their precursor ions. The extraction of neutral loss lines corresponding to the loss of phosphate under IRMPD fragmentation allows the selective identification of phosphopeptides. Resonance of the 10.6 µm infrared radiation with the vibrational modes of the phosphate functional group produced efficient absorption and high cleavage coverage of the phosphopeptides at much lower irradiation fluence than for nonphosphorylated peptides improving discrimination. Additionally, the localization of the phosphate group was determined.