A new ion mobility/time-of-flight mass spectrometer employing a high-pressure MALDI source has been designed and tested. The prototype instrument operates at a source/drift cell pressure of 1-10 Torr helium, resulting in a mobility resolution of approximately 25. A small time-of-flight mass spectrometer (20 cm) with a mass resolution of up to 200 has been attached to the drift cell to identify (in terms of mass-to-charge ratio) the separated ions. A simple tripeptide mixture has been separated in the drift tube and mass identified as singly protonated species. The ability to separate peptide mixtures, e.g., tryptic digest of a protein, is illustrated and compared to results obtained on a high-vacuum time-of-flight instrument.

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Ion mobility-mass spectrometry (IM-MS) data is interpreted as evidence that gas-phase bradykinin fragment 1-5 (BK1-5, RPPGF) [M ϩ H] ϩ ions exist as three distinct structural forms, and the relative abundances of the structural forms depend on the solvent used to prepare the matrix-assisted laser desorption ionization (MALDI) samples. Samples prepared from organic rich solvents (90% methanol/10% water) yield ions having an ion mobility arrival-time distribution (ATD) that is dominated by a single peak; conversely, samples prepared using mostly aqueous solvents (10% methanol/90% water) yield an ATD composed of three distinct peaks. The BK1-5 [M ϩ H] ϩ ions were also studied by gas-phase hydrogen/deuterium (H/D) exchange ion-molecule reactions and this data supports our interpretation of the IM-MS data. Plausible structures for BK1-5 ions were generated by molecular dynamics (MD). Candidate MD-generated structures correlated to measured cross-sections suggest a compact conformer containing a ␤-turn whereas a more extended, open form does not contain such an interaction. This study illustrates the importance of intra-molecular interactions in the stabilization of the gas-phase ions, and these results clearly illustrate that solution-phase parameters (i.e., MALDI sample preparation) greatly influence the structures of gas-phase ions. tudies of peptide and protein structure play a major role in modern molecular biophysics and drive development of new, more specific and sensitive techniques. Gas-phase techniques for sequencing peptides and proteins are well established, and significant progress has been made in developing similar techniques for characterization of secondary (2°) and tertiary (3°) structures of gas-phase peptides/proteins [1][2][3][4][5]. Such studies increase our understanding of intra-molecular interactions and drive development of sequencing algorithms, but the relevance of gas-phase structure, structure in the absence of solvent [6], to solution-phase structure is still an open issue. The most important role of solvent is charge stabilization, and in the gas phase, charge stabilization occurs by intramolecular interactions [7]. Rapid progress is being made in the development of techniques that allow selective solvation (in terms of number of solvent molecules) of gas-phase peptide/protein ions [8], and these studies may serve to further our understanding of how solvent affects structure and function. In addition, gas-phase studies could enhance our understanding of intra-molecular interactions (i.e., hydrogen bonding, van der Waals interactions and hydrophobic forces), as well as how such interactions influence conformations in the solution phase or low dielectric environments (i.e., gas phase, membranes, and/or multimeric complexes).Bradykinin (amino acid sequence RPPGFSPR) has been extensively studied using both experimental and computational techniques. Bradykinin is an excellent model peptide for structural studies because the presence of arginine (R) at both the N-and C-terminus can potent...

Peptide sequencing by surface-induced dissociation (SID) on a MALDI-ion mobility-orthogonal TOF mass spectrometer is demonstrated. SID of approximately 100-fmol amounts of model peptides HLGLAR (m/z 666.8), gramicidin S (m/z 1142.5), and bovine insulin b chain (m/z 3495.5) was accomplished using hydrocarbon-coated gold grids and approximately 20-eV collision energies. The current version of the instrument achieves a mobility resolution of approximately 20 and TOF mass resolution better than 200. Peptide sequences of four peptides from a tryptic digest of cytochrome c (approximately 1 pmol deposited) were obtained. The advantage of IM-SID-o-TOF-MS is that a single experiment can be used to simultaneously measure the molecular weights of the tryptic peptide fragments (e.g., peptide mass mapping) and partial sequence analysis, (e.g., real-time tandem mass spectrometry.)