BACKGROUND
Mass spectrometry is revolutionizing the study of complex molecules, and anticipated advances in biological chemistry and proteomics now hinge upon the central contributions of mass spectrometric methods.1 Tandem MS is a critical tool in these developments, used to establish protein sequences, reaction kinetics, related cross-sections, parent ion stability, and parent-fragment ion correlations. Despite its successes, obstacles remain to important future applications of tandem MS. Among these are the lack of well-understood and consistent fragmentation methods to ensure complete and unambiguous parent/fragment ion correlations and protein sequencing, the difficulties associated with characterizing a specific complex molecule in a mixture, and the need for improved sensitivity and expanded dynamic range. Furthermore, for the quantification and sequencing of proteins, analysis of protein-ligand interactions, and the interpretation of post-translational modification - issues central to the expanding field of proteomics - the nature of the fragmentation processes are of particular importance.
Studies to date have largely relied on collision-induced vibrational excitation as a fragmentation technique. However, information related to the presence and locations of post-translational modifications (PTMs) are generally lost given the statistical nature of this process (i.e., always the weakest bonds are broken) and the labile nature of these functionalities. The development of a means to employ alternative, “non-ergodic” fragmentation techniques such as vacuum ultraviolet (VUV) laser photo-fragmentation and electron capture2 or electron-transfer3 dissociation (ECD, ETD), is a key aspect of our new approach. These processes, although poorly understood, show great promise in “top-down” protein sequencing and preserving PTMs. ECD and ETD dissociation are very promising non-ergodic means of fragmenting proteins that have been shown to preserve many delicate post-translational protein modifications. Broad use of ECD has not been possible, however, owing to its limitation to expensive and complex Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. The reasons for this are both that the interaction time with the electrons or anions must be long and the energy must be very low for the electron capture efficiency to be adequate. These are difficult conditions to achieve in conventional ion traps. In our approach, these obstacles are overcome using coupled cation and electron/anion electrostatic traps. In addition, VUV laser fragmentation can be readily incorporated in our design, and this is yet another promising non-ergodic fragmentation strategy. The novel coupled ion trap design, described in the accompanying pages, is the heart of this approach.
1. J. R. Yates, J. Mass Spec. 33, 1 (1998).
2. J. P. Gaut, J. Byun, H. D. Tran, and J. W. Heinecke, Anal. Biochem. 300, 252 (2002).
3. D. M. Good, W. Wirtala, G. C. McAlister and J. J. Coon, Mol. Cell. Proteom. 6.11, 1942 (2007).