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ANALYTICAL CURRENTS
Capturing electrons for MALDI-TOFMS DNA sequencing Even with automated electrophoretic DNA sequencing, elucidating a single sequence can require several hours. On the other hand, a complete matrix-assisted laser desorption/ionization time-of-flight (MAIDI-TOF) mass spectrum can be collected in less than 1 ms, not counting data manipulation. However, DNA sequencing by MALDI-TOFMS is limited by a dramatic drop in sensitivity for sequences longer than 30 nucleotides. Before DNA can be detected by MALDI-MS, it must be desorbed and ionized. Improving the efficiency of the processes would help in detecting larger DNA species. Efficient desorption has been demonstrated for double-stranded DNA molecules containing 622 bp and single-stranded DNA containing an average of 400 nucleotides, but efficient methods of ionization are still needed. Gregory Hurst and co-workers at Oak Ridge National Laboratory have investigated whether electrophores attached to oligonucleotide primers could enhance ioniza-
tion by using electron capture rather than proton transfer as the means of ionization. They attached four different electrophores (/>-nitrophenyl-isothiocyanate, AT-succinirnidyllpentafluorobenzoate, JV-succinimidyl-4-carboxy-9-fluorenone, and A^succinimidyl-3-nitrobenzoate) to the 5'-terminus of 17-base, single-stranded DNA sequencing primers. Little or no fragmentation resulted from the loss of the electrophore labels, which would hav< led to a peak at a mass-to-charge ratio corresponding to that of the unlabeled primer. These results demonstrated that the electrophore-labeled primers could be successfully synthesized, that the electrophores remain attached to the primer throughout the MALDI-MS preparation and desorption steps and that picomole measurements are possible But despite the encouraging results they did not observe an increased sensitivity of the MS measurement and plan to investigate the possibility of instrumental modifications that favor electron capture ionization such as combining delayed extraction with the flooding of the TOF reeion with low-enercrv electrons ( J Mass Ibertrnm IQQfi 97 fifi1-fi8">
The image of corrosion
which provides a 7-cm _1 bandpass with a 50-um entrance slit and a 590-grooves/mm grating.
Would anyone want to use an imaging spectrometer with poor spectral and spatial resolution? Of course not, which is why tunable filters such as the acoustooptic tunable filter and the liquid crystal tunable filter (LCTF) are not widely used despite being solid-state devices with no moving parts. Why settle for the 50-cm"1 spectral bandpass of an AOTF when a dispersive monochromator can provide much better? Well, the tide may be turning for tunable filters. Patrick Treado and co-workers at the University of Pittsburgh and Cambridge Research & Instrumentation have incorporated a Lyot-type LCTF with diffraction-limited spatial resolution and a 7.6-cnT1 spectral bandpass into an infinity-corrected optical microscope for Raman imaging. The LCTF provided spectra comparable to those obtained with a 0.5-m dispersive monochromator,
MALDI negative-ion mass spectra of electrophore -labeled primers. (a) p-nitrobenzene derivative, 2pmol on probe tip, (b) pentafluorobenzoyl derivative, 7 pmol on probe tip. The dashed vertical line is the mass-to-charge ratio of the underivatized primer. (Adapted with permission of John Wiley & Sons.)
They used the microscope to obtain images of a 4.3-um standard resolution target, of 45-um and 2-um polystyrene microspheres, and of a corrosion products test sample comprising KN03,Ti02,andW03. Brightfield images of the 2-um polystyrene microspheres contain Airy rings, which indicate that the spatial resolution of the LCTF is diffraction limited. The polymer and corrosion products model systems indicate the potential aoolicability of Raman imaging with LCTFs to materials research The wide acceptance angle of LCTFs makes them adaptable to Composite image of a model corrosion products sample. macroscopic imaging as KN03 (blue) was imaged at 1057 cm~1, Ti02 (red) imaged at well (AM ^fiprtrmr 1 1 604 cm~ , and W03 (yellow) imaged at 797 cm~ . (Courtesy IQQfi Vt SOS-Ill of Patrick Treado.) Analytical Chemistry News & Features, September 1, 1996 5 2 3 A
