Infrared matrix-assisted laser desorption/ionization of polycyclic aromatic hydrocarbons with a sulfolane matrix.

Infrared matrix-assisted laser desorption/ionization (IR-MALDI) of the polyaromatic hydrocarbons (PAHs) anthracene, benzo[a]pyrene, and dibenz[a,h]anthracene was performed using a 10.6-microm CO2 laser and a liquid matrix. Sulfolane (tetrahydrothiophene 1,1-dioxide) was found to be an effective matrix for PAH ionization: mass spectra obtained with a sulfolane matrix contain an intense molecular ion peak; interference from PAH fragment and matrix peaks is negligible in all cases. The main limitation of the sulfolane matrix is sample evaporation after 3 to 5 min in vacuum. This sample lifetime can be increased to between 15 and 30 min using a 2:1 (v/v) mixture of sulfolane and glycerol, but the resulting spectra have greater matrix interference and decreased shot-to-shot signal stability.

Selective degradation of oxidized calmodulin by the 20 S proteasome.

We have investigated the mechanisms that target oxidized calmodulin for degradation by the proteasome. After methionine oxidation within calmodulin, rates of degradation by the 20 S proteasome are substantially enhanced. Mass spectrometry was used to identify the time course of the proteolytic fragments released from the proteasome. Oxidized calmodulin is initially degraded into large proteolytic fragments that are released from the proteasome and subsequently degraded into small peptides that vary in size from 6 to 12 amino acids. To investigate the molecular determinants that result in the selective degradation of oxidized calmodulin, we used circular dichroism and fluorescence spectroscopy to assess oxidant-induced structural changes. There is a linear correlation between decreases in secondary structure and the rate of degradation. Calcium binding or the repair of oxidized calmodulin by methionine sulfoxide reductase induces comparable changes in alpha-helical content and rates of degradation. In contrast, alterations in the surface hydrophobicity of oxidized calmodulin do not alter the rate of degradation by the proteasome, indicating that changes in surface hydrophobicity do not necessarily lead to enhanced proteolytic susceptibility. These results suggest that decreases in secondary structure expose proteolytically sensitive sites in oxidized calmodulin that are cleaved by the proteasome in a nonprocessive manner.

A rotating ball inlet for on-line MALDI mass spectrometry.

The rotating ball inlet (ROBIN) is presented in a new design for on-line matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). This method uses a capillary to deliver a matrix and analyte solution to the surface of a rotating ball upon which MALDI is carried out. The ball is in contact with a polymer gasket surrounding the capillary. Sample adhering to the surface of the ball is dragged past the gasket into the vacuum of the mass spectrometer where it is irradiated by a pulsed UV laser, and the resulting ions are mass-separated in a linear time-of-flight mass spectrometer. The mechanical sample introduction prevents clogging of the vacuum interface by matrix crystals or frozen solvent. Preliminary results from flow injection analysis (FIA) suggest that the new interface does not introduce a significant peak-tailing or memory effect. The system is capable of 20-30 h of continuous operation with a flow rate of 2 microL/min before cleaning of the ball is needed. With the prototype inlet, concentration detection limits are at the low micromolar level.

337 nm matrix-assisted laser desorption/ionization of single aerosol particles.

Matrix-assisted laser desorption/ionization (MALDI) mass spectra were obtained from single particles injected directly into a time-of-flight mass spectrometer. Aerosol particles were generated at atmospheric pressure using a piezoelectric single-particle generator or a pneumatic nebulizer and introduced into the mass spectrometer through a series of narrow-bore tubes. Particles were detected by light scattering that was used to trigger a 337 nm pulsed nitrogen laser and the ions produced by laser desorption were mass separated in a two-stage reflectron time-of-flight mass spectrometer. MALDI mass spectra of single particles containing bradykinin, angiotensin II, gramicidin S, vitamin B(12) or gramicidin D were obtained at mass resolutions greater than 400 FWHM. For the piezoelectric particle generator, the efficiency of particle delivery was estimated to be approximately 0.02%, and 50 pmol of sample were consumed for each mass spectrum. For the pneumatic nebulizer, mass spectra could be obtained from single particles containing less than 100 amol of analyte, although the sample consumption for a typical mass spectrum was over 400 pmol.

A Laminar Flow Nebulizer for Aerosol MALDI.

A laminar flow nebulizer was developed for use with aerosol matrix-assisted laser desorption/ionization mass spectrometry. The nebulizer consists of a glass pneumatic nebulizer combined with a laminar flow tube. Particles formed at the pneumatic nebulizer are entrained in an auxiliary gas stream that dries the particles in the laminar flow tube. The mass range of the reflectron time-of-flight mass spectrometer has been extended to greater than 5000 Da: a mass spectrum of bovine insulin is reported at a mass resolution of 100. Mass spectra of bovine insulin and the peptides bradykinin and angiotensin II contain features resulting from prompt analyte dissociation prior to ion acceleration.

DNA sequencing by mass spectrometry.

Mass spectrometry has the potential to replace gel electrophoresis for fast DNA sequencing. In this tutorial paper, it is discussed how far mass spectrometry of DNA has come since the advent of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) and how much remains to be done before mass spectrometry will be a useful tool for DNA sequencing. A brief description of MALDI and ESI mass spectrometric analysis of DNA is presented along with the different strategies for DNA sequencing using mass spectrometry. These include mass spectrometric analysis to replace gel separation in Sanger sequencing, enzymatic ladder sequencing and sequencing by gas-phase fragmentation. The future role of mass spectrometry in DNA sequencing in the Human Genome Project and beyond is also discussed.

Aerosol MALDI with a Reflectron Time-of-Flight Mass Spectrometer.

Aerosol matrix-assisted laser desorption/ionization (MALDI) has been combined with a reflectron time-of-flight mass spectrometer for improved mass resolution. A methanol solution of matrix and analyte was sprayed directly into a reflectron time-of-flight mass spectrometer, and the aerosol particles were irradiated and ionized with a frequency-tripled Nd:YAG laser at 355 nm. Mass resolution of over 300 was observed for the peptides bradykinin, angiotensin II, and gramicidin D and for vitamin B(12). This represents a resolution enhancement of approximately 10-fold over that previously reported for aerosol MALDI with a linear time-of-flight instrument.

Aerosol matrix-assisted laser desorption ionization mass spectrometry.

A new method of liquid sample introduction for a time-of-flight mass spectrometer (TOF-MS) has been developed by applying the method of matrix-assisted laser desorption ionization to aerosols. Analyte biomolecules are dissolved in a methanol solvent along with a UVabsorbing matrix and formed into an aerosol with a pneumatic nebulizer. The aerosol particles are dried in a heated skimmer tube before ionization by pulsed 355-nm UV laser radiation. Mass analysis is achieved in a linear TOF-MS. Results for the ionization of bovine insulin (5733.5 Mw) are reported.

Reproducibility and quantitation of matrix-assisted laser desorption ionization mass spectrometry: effects of nitrocellulose on peptide ion yields.

We have observed that using nitrocellulose in sample preparation for matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry increases the yield of peptide [M+H]+ ions. Addition of nitrocellulose also provides improvement in sample-to-sample reproducibility of MALDI ion yield and improves the precision of peptide quantitation. Mass spectrometry and optical microscopy results suggest that the nitrocellulose modifies the crystallization of matrix/analyte solution to allow more even coverage over the sample surface.

Angiography of the lower extremity in atherosclerotic vascular disease. Current techniques.

With the vast array of technology available, detailed diagnostic information is now routinely obtained in patients with peripheral vascular disorders. In addition, percutaneous intervention and advanced surgical techniques now safely permit the patient to return to an active life, and in advanced vascular disease, the extremity can be salvaged when previously the only option was amputation. We feel strongly that the maximum amount of information about the arterial system should be obtained throughout the patient's management. In addition, these complex procedures require considerable experience and judgment, and our practice of ongoing communication with the vascular surgery staff during both the diagnostic and the therapeutic phases of the patient's treatment has been extremely beneficial.

Renal peripelvic lymphangiectasia: appearance at CT.

In a patient who had undergone emergency nephrectomy during exploratory surgery, a pathologic diagnosis of peripelvic lymphangiectasia was made. Subsequent computed tomographic examination of the remaining kidney showed large multilocular renal sinus cysts, renal capsular cysts, and regions of fluid attenuation surrounding the mesenteric vessels, findings consistent with peripelvic lymphangiectasia. A brief discussion of cystic disease of the renal sinus is presented.