Novel Hybrid Quadrupole-Multireflecting Time-of-Flight Mass Spectrometry System.

A novel mass spectrometry system is described here comprising a quadrupole-multireflecting time-of-flight design. The new multireflecting time-of-flight analyzer has an effective path length of 48 m and employs planar, gridless ion mirrors providing fourth-order energy focusing resulting in resolving power over 200 000 fwhm and sub-ppm mass accuracy. We show how these attributes are maintained with relatively fast acquisition speeds, setting the system apart from other high resolution mass spectrometers. We have integrated this new system into both liquid chromatography-mass spectrometry and mass spectrometry imaging workflows to demonstrate how the instrument characteristics are of benefit to these applications.

Combination of Edman degradation of peptides with liquid chromatography/mass spectrometry workflow for peptide identification in bottom-up proteomics.

RATIONALE: High-throughput methods of proteomics are essential for identification of proteins in a cell or tissue under certain conditions. Most of these methods require tandem mass spectrometry (MS/MS). A multidimensional approach including predictive chromatography and partial chemical degradation could be a valuable alternative and/or addition to MS/MS. METHODS: In the proposed strategy peptides are identified in a three-dimensional (3D) search space consisting of retention time (RT), mass, and reduced mass after one-step partial Edman degradation. The strategy was evaluated in silico for two databases: baker's yeast and human proteins. Rates of unambiguous identifications were estimated for mass accuracies from 0.001 to 0.05 Da and RT prediction accuracies from 0.1 to 5 min. Rates of Edman reactions were measured for test peptides. RESULTS: A 3D description of proteolytic peptides allowing unambiguous identification without employing MS/MS of up to 95% and 80% of tryptic peptides from the yeast and human proteomes, respectively, was considered. Further extension of the search space to a four-dimensional one by incorporating the second N-terminal amino acid residue as the fourth dimension was also considered and was shown to result in up to 90% of human peptides being identified unambiguously. CONCLUSIONS: The proposed 3D search space can be a useful alternative to MS/MS-based peptide identification approach. Experimental implementations of the proposed method within the on-line liquid chromatography/mass spectrometry (LC/MS) and off-line matrix-assisted laser desorption/ionization (MALDI) workflows are in progress.