Dramatic progress has been made in the development of new mass spectrometry techniques for the analysis of peptides and small proteins. What is remarkable about these techniques is that they produce mass spectra that have large peaks for the protonated, or multiply protonated, molecular ions. These peaks are very useful for determining the molecular weight of a peptide sample, but often there are not enough fragment peaks to determine the amino acid sequence. One approach to solving this problem is to use a tandem mass spectrometer to fragment the parent ions by passing them through a gas collision cell. This technique, called collision- induced dissociation (CID), works well for low mass peptides, but is less efficient for high mass ions. In this grant application, we propose to develop laser photodissociation as an alternative to CID for rapidly sequencing peptides and determining the structures of modified peptides.
The specific aims of our research are (1) to improve the mass resolution and sensitivity of our quadrupole-Fourier transform mass spectrometer (QFTMS) for the analysis of peptides and small proteins, (2) to obtain a fundamental understanding of the mechanism of UV laser photodissociation of peptides, and (3) to study the slow metastable decay of peptide ions formed under different conditions. For these studies a specially-designed Fourier transform mass spectrometer with an external ion source will be used. With this instrument a sample is placed in the external ion source and ionized by fast atom bombardment (FAB). The ions are then extracted from the source region and focused into a narrow beam by a 1.2 m-long quadrupole ion guide. The ion guide injects the ions axially into a 6-T superconducting magnet where they are trapped in a FTMS analyzer cell. Photodissociation experiments are performed by using a series of radiofrequency ejection pulses to isolate a particular parent mass, and then a laser pulse is fired to dissociate the ions. This method has great potential for elucidating the structures of high mass biomolecules because the trapped ions can be irradiated with many laser pulses if necessary, to cause dissociation. The third area mentioned above, slow metastable decay of high mass ions, is suggested by our recent observations that protonated molecular ions of gramicidin S, substance P, and several other peptides decompose slowly while they are trapped in the FTMS analyzer cell. The rate of metastable decay can be measured by monitoring the relative abundance of the ions as a function of storage time.
| McIver Jr, R T; Li, Y; Hunter, R L (1994) Matrix-assisted laser desorption/ionization with an external ion source Fourier-transform mass spectrometer. Rapid Commun Mass Spectrom 8:237-41 |
| McIver Jr, R T; Li, Y; Hunter, R L (1994) High-resolution laser desorption mass spectrometry of peptides and small proteins. Proc Natl Acad Sci U S A 91:4801-5 |
| Li, Y; McIver Jr, R T; Hunter, R L (1994) High-accuracy molecular mass determination for peptides and proteins by Fourier transform mass spectrometry. Anal Chem 66:2077-83 |
| Li, Y; McIver Jr, R T (1994) Detection limits for matrix-assisted laser desorption of polypeptides with an external ion source Fourier-transform mass spectrometer. Rapid Commun Mass Spectrom 8:743-9 |
