大家谁有在美国举行的52界ASMS会上刊登的论文？

Time or Slot: 206

Identification and Characterization of the Major forms of Glutathione S-Transferase in Mouse Liver

Timothy K Nadler; Barrie G Wagenfeld; Robert J Lotti; Carlton H Paul; George J Vella;
Applied Biosystems, Framingham, MA

IntroductionThere are eight classes of glutathione S-transferase (alpha, mu, pi, theta, sigma, kappa, zeta and omega) which play an important role in the detoxification pathways in liver. We have been able to uniquely identify four of these isoforms based on peptide mass finger-printing (PMF) from crude mouse liver homogenates after SDS-PAGE (mu, theta, pi and alpha), even though two of the isoforms co-migrate. Our goal is to assess the ability of MS based techniques to rapidly determine the relative abundances of these multiple isoforms from crude samples without the use of antibodies. These techniques could then potentially be used to study the effect of drug toxicity on the expression of the various transferases.
MethodsLivers from C57BL/6 mice were dissected and homogenized. The homogenate was then centrifuged and the supernatant was prepared for SDS-PAGE using the molecular scanner technique (Hochstrasser et al. ref 1&2). The peptides observed by the molecular scanner process were then used to identify the proteins originally in the tissue homogenate using the ChemApplex peptide mass fingerprinting program (ref 3). Mouse tissues were also sliced and placed on the membrane stack of the molecular scanner technique in the place normally reserved for the SDS-PAGE gel. In this way, proteins from the tissue slice were electroblotted out of the tissue through an immobilized trypsin membrane and finally onto a capture membrane. Tissue images were reconstructed based on where specific masses were observed.
Preliminary ResultsFrom crude mouse liver homogenates, four distinct glutathione S-transferase classes, alpha, mu, pi and theta, were identified using the molecular scanner technique. The sums of the MS intensities of the identified proteins were plotted by the degree of migration in the 1-D gel lane and are visualized using a Visual Basic® program. The migration distances observed using this visualization tool were consistent with the molecular weights of the GSTs, ranging from 23-29 KDa. These images resemble Western blots but without the need for antibodies and images for any protein identified in the experiment and are not limited to the GSTs. Further, multiple proteins within a single band can be identified and visualized individually. Over 100 proteins, in addition to the GSTs, were identified in the crude mouse liver homogenate. GST-mu was identified as the major form of GST observed in the liver tissue section with MH+ peptide masses of 1287.5, 1479.7, and 1749.8 m/z. References: (1) Bienvenut WV, Sanchez JC, Karmime A, Rouge V, Rose K, Binz PA, Hochstrasser DF. Anal Chem. 1999, 71(21):4800-4807. (2) Binz PA, Muller M, Walther D, Bienvenut WV, Gras R, Hoogland C, Bouchet G, Gasteiger E, Fabbretti R, Gay S, Palagi P, Wilkins MR, Rouge V, Tonella L, Paesano S, Rossellat G, Karmime A, Bairoch A, Sanchez JC, Appel RD, Hochstrasser DF. Anal Chem. 1999, 71(21):4981-4988. (3) Parker KC, J Am Soc Mass Spectrom. 2002 Jan;13(1):22-39.

Time or Slot: 207

Imaging MALDI with an Orthogonal TOF Mass Spectrometer

Gamini Piyadasa; James R McNabb; Vic Spicer; Kenneth G Standing; Werner Ens;
University of Manitoba, Winnipeg, MB, Canada

IntroductionAn orthogonal-injection MALDI TOF instrument is well-suited for obtaining mass-selected 2d images from tissue sections because the source is decoupled from the mass measurement. The mass spectral quality is thus independent of variations in sample properties (such as thickness), and the target may be held at low voltage and in a modest vacuum. Moreover it allows greater flexibility for the incident laser optics and also allows the possibility to perform MS/MS measurements on selected peptides or proteins.
MethodsWe have constructed a new MALDI source for orthogonal TOF in which the ions are ejected perpendicular to the axis of the collisional cooling ion guide. This allows normal incidence for the desorbing laser, and much closer placement of the final focusing optic, both of which are essential for high-resolution imaging. Desorbed ions are drawn into the ion guide by gas flow. For the present experiments, a high-repetition rate Nd-Yag laser is coupled to a 10-micron optical fibre. The output is imaged one-to-one on the sample and rastered at a uniform rate to obtain an image. A continuous data log of flight times is acquired along with real-time markers to coordinate the position of the laser spot.
Preliminary ResultsThe source and software have been tested with a 500 lines-per-inch grid, coated with angiotensin and placed on a target coated with C60. Mass selected images for both species clearly show spatial resolution of 10 microns or better. With a 500 Hz repetition rate laser, the images can be acquired at 10 pixels per second, although with tissue samples, the rate will likely be determined by the abundance of the protein of interest. Preliminary images of small proteins up to m/z 4000 have been obtained at this rate from some plant tissues. Focusing the laser directly onto the target with a lens placed about 1 cm from the surface will test the limits of spatial resolution of imaging MALDI. This is expected to produce spot sizes less than 2 microns in diameter.

Time or Slot: 208

Laser Desorption/Ionization Time-of-Flight Mass Spectrometric Characterization of Plant Cuticular Wax by using Colloidal Silver Additive.

Chanan Sluszny; Edward S Yeung;
Iowa State University, Ames, IA

IntroductionLaser desorption ionization time-of-flight without a matrix can be used for analysis of various low-molecular-weight compounds, since these usually remain intact during laser irradiation. However, non-polar hydrocarbons (HCs) give no result under such conditions of ionization. It has been shown, however, that certain salts of transition metals, such as silver nitrate, when co-deposited on the HC sample, give cataionized species that can be analyzed in a mass-spectrometer. This method works well for HC-containing materials such as wax and non-polar lipids. It is, however, highly dependent on the amount of metal additive and on laser power. The goals of this study are to establish a simple and reproducible methodology for analysis of cuticular wax compounds within intact plant material.
MethodsLDI-TOF spectra were recorded using a Voyager-DE PRO instrument equipped with a N2 laser. Mass spectra were acquired in the reflector mode. Positive ions were subjected to a 20 kV accelerating potential. Typically, 100 laser pulses were acquired for each measurement. For the pure hydrocarbon compounds (HCs) a two-layer technique was used for sample preparation. Samples were dissolved in THF and dried on the probe. Then, a metal additive (salt, metal powder or colloidal suspension of silver) was deposited onto the sample and dried. Fresh plant material from Arabidopsis was used as target material for analysis of wax compounds. Samples were cut into 2 mm segment

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