An FT-IR alternative to the Pap smear The Papanicolaou smear test, or "Pap smear", is commonly used to screen for preinvasive human cervical cancer. Despite its widespread acceptance, the Pap smear has a sufficiently high false negative rate (20%) to prompt Donald McNaughton and colleagues at Monash University (Australia) and the University of Melbourne (Australia) to investigate the use of FT-IR spectroscopy as an alternative means of detecting cervical cancer. The method is based on observing changes in tissue biochemistry that must precede any morphological or symptomatic manifestations of the cancer. The researchers studied exfoliated cervical cells from 272 patients. Six spectra were re-
corded for each patient, and these were visually sorted into two types based on their profiles. Type 1 spectra exhibited a profile characteristic of normal epithelial cells, with intense glyocogen bands and a pronounced symmetric phosphate stretch. Type 2 spectra exhibited features suggestive of dysplastic or malignant transformation, with pronounced symmetric and asymmetric phosphate modes and a reduction in glycogen band intensity. Principal component analysis (PCA) using a reduced data matrix resulted in a score plot that showed general correlation with the visually categorized spectra. Strong correlation between spectra visually classified as abnormal and an abnormal biopsy indicates that visual inspection or subsequent PCA of cervical sample spectra is comparable to the Pap smear.
PM-NSOM of rhodamine 110 Near-field scanning optical microscopy (NSOM) beats the diffraction limit by providing two things smaller than the wavelength of light used—the size of the source and the distance from the source to the sample. Because visible light is used, contrast mechanisms such as fluorescence and refractive index changes are the most common. Paul F. Barbara and co-workers at the University of Minnesota have designed a new form of polarization-modulation (PM) NSOM that allows detailed characterization of optically anisotropic samples through simultaneous collection of images that give the magnitude and direction of the optical anisotropy. The linearly polarized light coupled into the NSOM probe is rotated through 180° at 2 kHz, with 2L combination of an electrooptic modulator and a quarter-wave plate. The anisotropic optical properties of the sample produce a polarization-dependent modulation in the intensity of near-field light coupled through the sample to the detector Using lock-in detection two images are recorded as the amplitude and phase of the polarization-dependent response For PM-NSOM imaging of samples that absorb the probe wavelength ("resonant PM-NSOM"), the amplitude image provides a measure of the local anisotropy of the extinction coefficient, and the phase image indicates the transition dipole orientation. These effects are demonstrated by imaging rhodamine 110 with the 514-nm line of an argon ion laser.
Spectra of malignant HeLa cells also display spectra similar to type 2 spectra in the 1300-950 cm-1 region. (Biospectroscopy 1996,2,143-53)
Representative spectra from type 1, type 2, and HeLa cells. (Adapted with permission of John Wiley & Sons.)
New strategy for assigning MS protein fragments One way to probe the structure and behavior of macromolecules such as polypeptides is with soft ionization MS techniques. However, identifying the fragment ions in the spectra can be a nightmare. For example, Christopher M. Dobson and colleagues from the University of Oxford (U.K.) and Bruker Instruments have looked at fragment ions produced during the collisioninduced dissociation (CID) of the well-characterized protein hen lysozyme. Exhaustive computer searching for the peak at 584.4 yields 96 fragments that have calculated m/z within 1000 ppm of this Vcduc unci 30 within 50 To overcome this problem, the researchers studied the CID-MS of uniformly isotopically labeled proteins that are normally prepared for NMR studies and used atom counting to determine elements of the ion fragment's empirical formula. Using the high resolving power Resonant PM-NSOM amplitude (top) and phase (bottom) images of rhodamine 110 crystals recorded at 514 nm.
PM-NSOM can also be used to image nonabsorbing samples ("nonresonant PMNSOM") provided that the refractive index of the sample is anisotropic in the near field. Samples can be imaged with shot-noise limited S/N and spatial resolution better than 100 nm. (J. .hys. Chem. 1996,100,13794-803)
CID-FTMS spectrum of hen lysozyme and distribution of natural abundance isotope peaks from six fragment ions.
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News of an FT mass spectrometer, the fragments of unlabeled lysozyme appear as multiplets because of the presence of naturally abundant isotopes. The fragment at 584.4 has isotopic peaks separated by 0.5 mass units indicating a charge of +2 and a mass of 1168.8 Da. Comparing the unlabeled spectrum with peaks in the corresponding spectra of isotopically labeled lysozymes provided a count of 53 carbons and 18 nitrogens for this fragment, and only one possibility with the correct carbon and nitrogen count is found within 50 ppm of the experimental m/z. With this general approach 20 of the 24 fragments found in the 500-2000 for the lysozvme were uniquely identified (/. Am Chem Soc 1199 118 7402-03)
Measuring ascorbic acid kinetics
Getting more information from MALDI-TOFMS Since its introduction a decade ago, matrix-assisted laser desorption/ionization MS (MALDI-MS) has grown in importance as a means to determine the molecular weight of biomolecules even in crude extracts or in peptide mixtures. However, MALDI is not only a technique for producing intact molecular ions. Depending on the matrix, irradiance, and ion source conditions, MALDI-MS experiments C3n also be used to generate ion fragments. For molecular weight screening, especially for mixtures of proteins, this fragmentation is undesirable and contributes to the degradation of mass resolution for larger proteins For smaller analytes on the other hand the fragmentation can facilitate the determination of the chemical structure a technique labeled postsource decay
Jianru Stahl-Zeng and Franz Hillenkamp of the University of Munster (Germany) and Michael Karas of the University of Frankfurt (Germany) have investigated metastable-ion analysis using a gridless reflection time-of-flight MS instrument, applying three different acceleration potentials: 5 kV, , 7V, and 10 kV. The gridless ion mirrors allow detectors with smaller active areas to be used compared with two-stage reflection instruments. The authors found that ion mirror tuning has a strong influence on instrument performance; they also describe a simple window-independent mass calibration function to calibrate fragment ions to a mass accuracy of 5 x 10"4. The full fragmentation pattern was observed even at low acceleration potential, but with a reduced mass resolution. Delayed ion extraction can be used to improve the resolution of precursor ion selection. {Eur. Mass Spectrom. 1996,2, 23-32)
Although ascorbic acid (vitamin C) is probably the most widely used vitamin supplement, there is no international consensus on daily requirements and the amount required for antioxidant protection. Nutritional surveys provide limited information, but tracer studies are necessary for detailed kinetic information. Adrian P. Izzard and colleagues at the Dunn Nutritional Laboratory (U.K.) have evaluated using stable isotopes rather than the commonly used radioactive 14C-labeled material for in vivo mcssurement. of ciscor* bic acid pharmacokinetics. They used electron impact GC/MS to measure the enrichment of ascorbic acid in plasma for a 24-h period after oral adPost source decay-MALDI of the peptide KA6-K in 2,5-DHB, with a wavelength of ministration of 13C-labeled material. (Be337 nm and prompt extraction. (Adapted with permission of IM Publications.) cause ascorbic acid absorption is known to be dose-dependent, with excess material excreted in the urine, the researchers limited the dose of 13C-labeled ascorbic acid to 50 mg, about the amount in one The ascorbic acid in the plasma samstudy, and that first-order kinetics may not large orange.) ples was derivatized with tetramethylsiadequately describe the metabolic clearlane (TMS), and the resulting tetra-TMS ance of ascorbic acid in humans. (J. Mass ascorbate was subjected to GC/MS using Spectrom. .199,31, 741-48) electron impact and positive-ion chemical ionization. Although they obtained only Crank up the pH partial chromatographic resolution, the researchers were able to use selected-ion Reversed-phase separation of basic commonitoring to quantitate the ascorbic acid pounds at pH < 3 often gives the best peak without interference from the glucose shape and column efficiency, although enantiomers that flank it. solute instability or band-spacing problems can result. Separation at pH 4-8 can Their results, although comparable to those reported in other published studies, solve these problems, but it also creates new ones. At pH 9 or above, separation indicate that the U.KL's recommended becomes very attractive because basic daily intake of 40 mg/day of ascorbic acid Typical chromatogram of sugars in plasma. compounds can be separated as free for normal adults may be insufficient to (Adapted wiih permission of John Wiley & bases, which minimizes interactions with maintain the body levels found in this Sons.) 596 A
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