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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-
librium resonance signal as a function of pH was not affected by steric strain between the protein and the dextran matrix. (Four glycine residues aligned linearly are approximately the same length as the radius of gyration of native DHFR.) They could monitor the course of protein denaturation induced by solutions of HC1 and potassium phosphate buffer over the pH range 0.12-7.80. However, they could not use more traditional denaturing agents such as urea and guanidium hydrochloride because the reagents themselves caused such a strong SPR response (the guanidium hydrochloride went off scale using their commercial instrument) that it would be all but impossible to distinguish contributions from changes in the protein conformation. The measurements were close to being in "real time", with only a lag of approximately 0.1 s between collecting the data and recording it on the computer. "This
prevents the very fastest conformational transitions from being monitored, but interface mixing when changing buffers is even more serious. We ignored the first 8 s of measurements following a pulse injection of a different buffer," says Sota. "Of course, like other methods for monitoring protein conformation, the measurements reflect the average status of many molecules rather than the real-time conformational status of any single molecule." The tethered protein displayed a threestate transition (native-state to acid-unfolded to globulelike conformation) with decreasing pH. The pH values of the transition points shifted to milder pHs than Sota had expected. They ruled out structural strain as the cause because the glycine-lengthened proteins exhibited a similar shift in transition points. They hypothesize instead that the pH shifts are caused by an environment within the matrix that differs from the bulk solu-
tion. They suggest that interactions with water molecules affect the protein's dielectric properties and, thus, its apparent refractive index. "I would hope for a firmer grasp of the mechanism by which protein conformational changes contribute to the SPR signal, be it the bound water effect we proposed or something else," says Sota. Sota believes that an SPR sensor specifically for protein folding should be possible with minor modifications. "The most important requirement would be to allow a greater change in the angle of least reflectance to be monitored by the instrument, which would allow the use of traditional denaturants such as guanidium hydrochloride and urea," Sota says. "I am less optimistic that an apparatus could be developed to monitor rapid thermal transitions. SPR measurements are very precise and delicate. We do not take measurements until the machine has equilibrated at any given temperature for a full hour." Celia Henry
Fluorescent polymer-based sensor for cAMP
fluorescent dye in the presence of cAMP (template molecule) and an initiator. Following polyBiosensors that rely on naturally occurring merization, the template molecule is extracted, recognition elements, such as antibodies leaving behind microcavand enzymes, offer great sensitivity and specificity; however, they suffer from stabil- ities that have threedimensional structures ity problems. As a result, there has been a complementary to cAMP. surge of interest in developing sensors with synthetic recognition sites, particu"There are two bindlarly those based on molecular imprinting ing functionalities for cAMP binds to fluorescent MIP techniques. William S. Powell, Petra Turke- cAMP in the polymer," witsch, and co-workers at McGill Universays Powell. "One is the sity (Canada) have found an approach that dye itself and the other is the HEMA We is too early to speculate why quenching is could lead to a new design of biomimetic speculate that both compounds bind with observed with the fluorescent MIP in the sensors in which a fluorescent dye is actucAMP." A large molar excess of HEMA presence of cAMP. They believe it may be ally imprinted into the recognition cavity. over cAMP was used in the imprinting pro- related to the restricted environment of the cess. "We suspect that the HEMA actually polymer, which would reduce the flexibility In their study, which appeared in the hydrogen bonds with the cAMP, creating a of the dye. "This is only a preliminary study,, May 15th issue of Analytical Chemistry emphasizes Turkewitsch. "We would have tt (p. 2025), molecular imprints that contain a cavity with enhanced recognition for the do a more in-depth photophysical study, fluorescent dye, £rans-4-[p-(N,N-dimethyl- ligand," explains Turkewitsch. looking at the fluorescence lifetimes, to deamino) styiyll-Af-vinylbenzylpyridinium In the presence of aqueous cAMP, the termine why we are getting quenching." chloride, are prepared against adenosine fluorescent MIP displays a quenching of 3':5'-cyclic monophosphate (cAMP), an fluorescence, whereas it shows almost no The authors investigate the effects of important second messenger in cells. The response to the presence of guanosine 3':5'- various concentrations of cAMP on the flufluorescent molecularly imprinted polymer cyclic monophosphate (cGMP), a comorescence of the polymers. Quenching of (MIP) serves as both the recognition elepound with a similar structure to cAMP. the fluorescence is observed with cAMP ment and the measuring element for The researchers found that without polyconcentrations in the range 100 nMcAMP. merization, the fluorescent dye displays an 100 uM. Above 100 uM cAMP, the eaccssienhancement in fluorescence in the presble sites on the polymer appear to be satuThe cAMP-imprinted polymer is preence of both cAMP and cGMP. In addition, rated. In all cases, a steady response is obpared by polymerization of a cross-linking served after approximately 30 min. The monomer (trimethylolpropane trimethacry- the nonpolymerized dye has a much lower affinity towards cAMP, says Powell. fluorescent MIP is stable enough to be late [TRIM]), afunctional monomer (2used more than once; however, so far the hydroxy methacrylate [HEMA]), and a According to Turkewitsch and Powell, it Analytical Chemistry News & Features, June 1, 1998 3 7 1 A
