Modifications to the newly developed tandem quadrupole Fourier-transform mass spectrometer have made it possible to record mass spectra on oligopeptides and small proteins in the mass range between 2 and 13 kDa.Methodology for the sequence analysis of proteins by tandem mass spectrometry has been under development in our laboratory for several years (1, 2). Presently this approach involves enzymatic and/or chemical degradation of the protein to a collection of peptides that are then fractionated by high-performance liquid chromatography. Each of 20 to 40 fractions containing as many as 10 to 15 peptides is then analyzed directly without further purification by a combination of liquid secondary-ion/collision-activated dissociation mass spectrometry on a triple-quadrupole instrument. Ions characteristic of the molecular mass of each peptide in a particular fraction are selected one after the other by the first quadrupole analyzer, these ions are dissociated by collision with argon atoms in the second quadrupole, and the masses of the resulting fragments are analyzed in the third quadrupole. The result is a collection of mass spectra characteristic of the amino acid sequence in each of the peptides produced in the enzymatic digest of the original protein. From an initial 10 nmol of 50-kDa protein, it is usually possible to obtain sequence information covering 25% to 60% of the sample in the 4 to 5 days required to do the above biochemical and instrumental manipulations. The major limitation of this approach has been the 1.8-kDa mass upper limit of our triple-quadrupole instrument. Maximum sequence information is obtained only when the protein under investigation is cleaved efficiently into peptides of molecular mass under this ceiling.To extend the methodology to mixtures of larger oligopeptides, we have recently constructed a tandem quadrupole Fourier-transform mass spectrometer (3, 4), similar to that described by McIver et al. (5). This instrument is equipped with a 7-T superconducting magnet and operates with a mass range in excess of 20 kDa. Fourier-transform instruments have tremendous potential for the analysis of large biological molecules (6-8), because (i) they record the masses of all ions in the spectrum simultaneously and do not have to be scanned while the sample is continuously consumed during ionization; (ii) they function as ion storage devices that permit accumulation of ions produced in low abundance from small amounts of sample; (iii) they facilitate direct analysis of oligopeptide mixtures by the doubleresonance technique (8); and (iv) they appear to be ideally suited for collision-activated dissociation (8) or laser photodissociation experiments (9) that produce fragment ions carrying sequence information from oligopeptide (M+H)+ ions.One disadvantage of Fourier-transform mass spectrometers is that they must be operated at a pressure less than 10-8 torr (1 torr = 133 Pa) in the analyzer to prevent collisions between ions and neutral gas molecules from interfering with the mass measurem...
Siegbahn, K.; Nording, C.; Fahlman, A; Nordberg, R., Hamrin, K.; Hedman, J.; Johansson, 0.; Bergmark, T.; Karlsson, S.; Llndgren, I.; Lindberg, B. "ESCA: Atomic Molecular, and Solid State Structure Studied by Means of Electron Spectroscopy"; Almqulst and Wlksells: Uppsab, 1967. Proctor, A.; Sherwood, P. M. A. Carbon 1983, 21, 53-59. Stutts, K. J.; Korach, P. M.; Kuhr, W. G.; Wightman, R. M. Anal.
The baste for mass analysis by Fourier transform km cyclotron resonance (FT-ICR) Is the motion of ions In a homogeneous magnetic field. Electric fields are used In FT-ICR to modify Ion motion (l.e., trapping and excitation fields). Ion Interaction with Inhomogeneous radio frequency and direct current (dc) electric fields complicates the Ion motion and leads to loss of both resolution and sensitivity. Modifying the geometry of the FT-ICR cell to produce uniform Ion acceleration and homogeneous dc trapping fields results in simplified frequency measurements and studies of Ion-molecule reactions.
A simple reversed phase high performance liquid chromatography (RP-HPLC) method was developed for the determination of putrescine, cadaverine and spermidine (a class of polyamines) in their benzoylated form from external known standards. In the optimization procedure, a number of parameters were examined: 1) Solvent used in the extraction of standard polyamines (diethyl ether versus chloroform); 2) Solvent used in the elution of the polyamine (methanol versus acetonitrile); 3) Mode of derivatization and extraction step(s) (derivatization and extraction performed together versus derivatization and extraction performed separately); and 4) Other instrumental parameters (such as UV detection wavelength, gradient profiles). The advantages of our method, relative to the standard Morgan method are: a) decreased chromatographic runtime, b) ease of preparation with good resolution, sensitivity, and reproducibility using a standard RP-HPLC method.
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