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Electrospray source
The source was coupled to a large-scale, C-shaped, reverse-geometry two-sector tandem mass spectrometer with a 0.754-m radius, 55° magnetic sector; 1-m radius, 81.5° electrostatic sector; and 6.5-m total flight path. The performance of the source was evaluated with gramicidin, bovine insulin, bovine ubiquitin, and cytochrome c. The source efficiently produced multiply charged ions of bovine ubiquitin and cytochrome c at accelerating potentials up to 10 kV. (Rev. Sci. Instrum. 1998, 69,1275-81)
Effective electrospray ionization sources for magnetic sector mass spectrometers must achieve relatively high mass resolution (>3000) and high ion current output (>5 pA) at an accelerating voltage of several kilovolts. Mikhail E. Belov and coworkers at the University of Warwick (U.K.) described an ESI source that can deliver a total ion current of as much as 20 pA at accelerating voltages up to 11 kV. The source design is based on a heated capillary tube and an assembly of two skimmers. The analytes were electrosprayed through a needle kept at a higher potential than that of a counter electrode and the capillary. The typical potential drop between the needle and the capillary was 3.5-4 kV. The droplets were desolvated by a combination of the elevated temperature in the capillary and the pump in the first vacuum region of the source Two other regions of the source were Schematic of the ESI source. 1. Electrospray needle; under vacuum. 2. counter electrode; 3. heated capillary tube; 4. first The ion current was optimized skimmer; 5. second skimmer; 6. extracting electrode; by adjusting the capillary temper- 7. first differential z-steering deflector; 8. y-focus ature and the potential bias belens; 9. differential y-steering deflector; 10. second tween the source components. A differential z-steering deflector; 11. z-focus lens. temperature range of 140-160 °C (Adapted with permission. Copyright 1998 American was used for the experiments. Institute of Physics.)
In search of a photoreversible chemosensor The ideal chemosensor would be selective, sensitive, and reversible. Jeffrey D. Winkler and co-workers at the University of Pennsylvania chose spiropyran as a starting point for cation sensors. They synthesized quinolinespiropyranindoline, which exhibited minimal fluorescence in ethanolic solution at 610 nm when excited with 550-nm radiation. Adding one equivalent of ZnCl2 to the solution and exciting at 572 nm (X.max xo the eompound when chelated with Zn2+) led to 14-fold increase in emission intensity at 610 nm. It was sensitive to 6.5 ppb Zn2+. The chelate did not release the cation when irradiated; however, the metal could be released by adding one equivalent of nitriloacetic acid. Still in search of a chemosensortiiatwas photoreversible, they synthesized nitroquinolinespiropyran. Because the compound exhibited strong absorbance in ethanolic solution, they used less polar solvents. The 370 A
fluorescence enhancement by one equivalent of ZnCl2 was less pronounced (ninefold) than the other compound, and thus the molecule was less sensitive (30 ppb Zn2+)) However, the chelation was —60% reversible by irradiation with visible light. The regenerated spiropyran was fully functional through 10 cycles. (J. .m. Chem. Soc. o998,120, 3237-42)
A photoreversible chemosensor. The fluorescence emission spectra of nitroquinolinespiropyran in 10~s M benzene at room temperature (A) before adding Zn2*; (B) after adding 1 equivalent ofZnCI2; and (C) after 30 s of irradiation with visible light.
Analytical Chemistry News & Features, June 1, 1998
SCIENCE
As the conformation changes Most methods for monitoring protein conformation require that the protein be free in solution, but, in real situations, proteins are more likely to be immobilized on a surface or embedded in a membrane. The ability to monitor conformational changes in immobilized proteins could allow the development of novel sensors, and surface plasmon resonance (SPR) could be the ideal method. Hiroyuki Sota and Yukio Hasegawa of Amersham Pharmacia Biotech and Masahiro Iwakura of the National Institute of Bioscience and Human Technology (both in Japan) describe the use of SPR to detect conformational changes in immobilized proteins. Their work appeared in the May 15th issue of Analytical Chemistry (p. 2019). A surface plasmon is an electromagnetic wave that propagates along the interface between a metal and a dielectric. Under conditions of total internal reflection, light can be coupled into surface plasmon modes, causing a decrease in the reflectivity. The SPR signal is a function of the apparent refractive index, which is determined by the mass and the dielectric properties of the material. SPR has primarily been used in the past to monitor protein mass changes at a surface, but because protein folding states affect the dielectric properties SPR should be able to monitor changes in the folding states. According to Sota, SPR has several advantages relative to other techniques for monitoring the conformation of immobilized proteins. First, the technique allows— even requires—protein immobilization, thus eliminating the problems caused by aggregation when proteins denature. Second, only a small amount of the protein is required and that amount can be used for repeated measurements (if the protein can renature spontaneously). In this work, Sota and his co-workers followed the acid denaturation of matrixbound Escherichia coli dihydrofolate teductase (DHFR) with SPR The protein was attached via a disulfide linkage to a carboxymethyldextran layer. To ensure that only one attachment site was possible, they replaced the two naturally occurring cysteine residues in the protein with alanine (Cys85) and serine (Cysl52). They also attached additional glycine residues before the terminal cysteine to verify that the equi-
