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releasedfromcells activated with recombinant mouse interferon-y and lipopolysaccharide. The sensor's limit of detection is 1 uM NO. The sGC-based sensor does not respond to common interferents, including nitrate, nitrite, and oxygen. However, the presence of glutathione was found to decrease its sensitivity. "The [glutathione interference] problem is unique to guanylate cyclase," says Barker. Cytochrome c'-based NO sensors are not affected by glutathione. Because glutathione is produced by macrophages, the sGC-based sensors had to be calibrated in a phosphate buffer containing the same amount of glutathione present in the cellular environment of interest. The heme domain and sGC are both relatively unstable proteins and, therefore, denature at high temperatures. "That is where the cytochrome c'-based sensors have an advantage—they are much more stable," says Barker. The sGC-based sensors can be used at room temperature for several hours, but they cannot be stored at room temperature overnight. However, sensors stored at 0-4 °C remain stable for several days. "They're like a lot of biological materials; you'd better keep them in the fridge," remarks Kopelman. Britt Erickson
that serves as an initial screen is performed with the porogen solvent, which, in this case, is methylene chloride. A MIP is most likely to bind to a template in the porogen solvent. Therefore, complete release of the template into the porogen solvent is a good indicator that the MIP is no good. "A rapid and quantitative release of template when washing the polymers in the porogenic solvent is a good indicator for absence of high affinity imprinted sites," says Sellergren. "On the other hand, retention of template is no evidence for their presence. However, a quantitative release ecn be used aa s ariterion for discarding materials, thus saving time in the further screening for selectivity." To test their assumption that release of the template into the porogen solvent means that the MIP is not selective, Sellergren and Lanza screened all of the MIPs— even the ones that had completely released the template in the initial screen— for selectivity for rebinding of terbutylazine (a triazine herbicide that had been used as the template molecule). The initial release test indicated that only polymers made with (trifluoromethyl) acrylic acid (TFM) and methacrylic acid (MAA) should be expected to show an enhanced affinity for the template molecule in methylene chloride. The results of the rebinding experiments were as they expected. "Only the polymers that significantly retained the Rapid MIPs template in the release test showed selective rebinding proDerties " savs Sellergren screening "Thus a complete release in the porogenic Selectivity is the word for molecularly imsolvent seems to be a valid criteri[on] to printed polymers (MIPs). Unfortunately, exclude materials" many factors involved in the synthesis of MIPs ultimately affect their recognition Polymers made with MAA as the funcproperties—the type and concentration of tional monomer rhowed the highest selecthe functional monomer, the cross-linking tivity for rhe template. Interestingly, , fullmonomer, the solvent (porogen), and the scale version of the MAA polymer, which template, as well as the experimental condi- was used as a chromatographic stationary tions. Synthesizing new MIPs or optimizing phase, displayed the highest capacity faca given MIP requires systematically varytor for atrazine in acetonitrile (k' = 18) that ing these factors and analyzing the resulthas been reported under similar condiing materials. tions. The retention of atrazine waa ssgnificantly higher with a polymer imprinted That's where Borje Sellergren and with terbutylazine than with a polymer Francesca Lanza of Johannes Gutenberg imprinted with atrazine. Sellergren susUniversity Mainz (Germany) come in. like pects that this is due to the difference in Toshifumi Takeuchi and his co-workers at basicity and hydrophobicity of the temHiroshima City University Qapan) (Anal plates and because the terbutylazine site Chem. 1999, 71, 285-90), Sellergren nnd can be expected to be sterically capable of Lanza have developed a method for rapidly accommodating atrazine. synthesizing and screening "mini" MIPs, which have been produced in a scaledAccording to Sellergren, the most imdown synthetic process (Anal. Chem. portant outcome of this work is the 1999, 71,2092-96)) However, ,n Selleramount oftimeit will save in synthesizing gren and Lanza's method, the washing step and optimizing MIPs. Although only six 374 A
Analytical Chemistry News & Features, June 1, 1999
MIPs were synthesized in this work, the method should be applicable to screening large numbers of MIPs. "There is no doubt that the screening methods developed by us and [Takeuchi's group] will bring significant savings in terms of time and reagents in the search for MIPs with desired performance," says Sellergren. Sellergren says they still have important steps to consider. "It is important to demonstrate the usefulness of the method in the imprinting of difficult templates where the present established protocol fails to produce MIPs with the desired recognition properties," he says. "Furthermore, because the consumption of template is typically a few milligrams, the imprinting of more precious templates can now be systematically assessed." In addition, he says it is important, in many cases, to find MIPs that recognize analytes in water or to find materials with higher sample load capacities or improved mass transfer properties. "In view of the large number of options it be of interest to find ways to further miniaturize the process" he says. Celia Henry NEWS FROM ABRF '99
AB
The Association of Biomolecular Resource Facilities
RF
Research •Trchnokig) • (.'on mm nival ton • Kdttrnlhm
ElizabethZubritskyreports from Durham, NC.
N e w biological standards considered Despite the enormous growth in bioanalytical techniques, the sole biological standard is still bovine serum albumin (BSA). However, the quality and compliance committee of the Association of Biomolecular Resource Facilities (ABRF) hopes to change that. The committee is working on plans for additional biological standards—peptides, proteins, and possibly other molecules—according to a report given by Alan J. Smith, chairman of the committee's working group for peptide standards. At this point, the committee has spoken to the National Institute of Standards and Technology (NIST) about developing new biological standards, but no formal ar-
rangement has been made, Smith said. However, one possibility is that ABRF would manufacture additional standards with assistance from NIST. Smith reported that the committee had selected three possible peptide standards with masses in the 1290- to 2950-Da range. All of them have been synthesized, according to Beth Fowler, who heads the committee. She said the peptides are intended to be amenable to MS/MS sequencing by MALDI and Edman degradation. The peptides vary in charge and hydrophobicity to make them useful for a variety of separation techniques, and they have sites suitable for cleavage by several proteases. Committee members and a few other researchers will characterize the peptides, she added and once that information has been collected the committee intends to submit a report to NIST. Similar plans for protein standards are under way but may take longer. Fowler said stability issues may make the process of selecting protein standards more time consuming. In addition, it may be necessary to select a set of proteins to cover techniques from protein sequencing to macromolecular interactions, she said. The committee is still looking into the need for carbohydrate and nucleic acids standards and is seeking comments about those possibilities and about peptide and protein standards, Fowler said. If you would like to offer suggestions or comments, please see the ABRF Web site (http://www.abrf.org) or contact Fowler at
[email protected].
Virtual electrophoresis
consistent preparation of the gel has proven to be "an art form", Loo added. Nevertheless, in one example with 443 predicted proteins, Loo said she could find 219 proteins (-50%) in the pH range of 6.1-6.6. By comparison, traditional 2-D electrophoresis with silver staining found 123 proteins (-28%). Similarly, in an example with 331 theoretical proteins, Loo found 260 (-79%) in the pH range 5.7-6.0, compared with 100 (—30%) found with silver staining. Although Loo said she was unsure if the method could be used for quantitation, she said it might be helpful for identifying modifications made to proteins. .n addition, ,ecause this method uses MS, it offers higher sensitivity and mass accuracy and the ability to study low molecular weight proteins, which sometimes get lost in traditional 2-D electrophoresis, Loo said.
Two-dimensional (2-D) gel electrophoresis has gone high-tech. "Virtual" 2-D electrophoresis has arrived. Developed by Philip Andrews at the University of Michigan and Rachel Loo and colleagues at Parke-Davis Pharmaceutical Research, the new method begins with 1-D isoelectric focusing separation. However, instead of a second round of electrophoresis, the pH-gradient gel is washed and soaked in a MALDI matrix solution. Then the gel is dried, and a 4-cm section is loaded onto the MALDI plate. After the researchers have acquired a spectrum from each spot on the gel, a computer translates the information into a 2-D gel format. Loo said the method was tested on an extract from E. coli, and proteins ranging from 20,000-100,000 Da were seen. In theory, with a molecular weight accuracy of 0.1% and a pi accuracy of 0.05 pH units, 92% oo the E. coli proteins could be identified, she said. However, in practice, the performance is not that high because the molecular weight accuracy is closer to 0.2%, ,nd 0.00 pH units is too optimistic. An- "Virtual" 2-D gel that shows E. coli proteins with other limiting factor is that approximate pis between 6.5 and 6.1
NEWS FROM THE ACS NATIONAL MEETING Celia Henry reports from Anaheim, CA.
Putting ion traps to the test There's been a lot of talk about ion traps in the past few years. Are these instruments up to taking over the applications usually reserved for other types of mass spectrometers? The results presented by Dana Barr of the Centers for Disease Control and Prevention (CDC) indicate that the answer is sometimes "yes," sometimes "no." At CDC, the rationale for choosing a particular mass spectrometer has historically not been defined, says Barr. In an effort to increase her laboratory's flexibility and trim the cost of analysis, she eval-
uated a quadrupole ion trap head-to-head with triple quadrupoles for urine samples and high-resolution (HR) mass spectrometers (i.e., magnetic sectors) for serum samples. The goal was to reduce the cost of analysis but to retain the sensitivity, selectivity, and precision of the existing method. For the high-resolution analysis, the analyte was methyl eugenol, an allyl benzene flavoring found in a wide variety of products, including baked goods and cigarettes. Although it is structurally similar to known carcinogens, no human pharmacokinetic data for methyl eugenol is available. The HRMS turned out to be 10-fold more sensitive and much more precise than the ion trap for the analysis of methyl
eugenol. Barr says that they will not be using an ion trap for the analyses they have traditionally done with HRMS. They compared the ion trap and the triple quadrupole for the analysis of chlorpyrifos, an organophosphate insecticide. With 25 pg injected on the column, the detection limit was approximately 0.9 ppb in 5 mL urine. They found that the ion trap mass spectrometer was less sensitive than the triple quadrupole, but that the sensitivity was adequate for human samples. In addition, the precision obtained with the ion trap was comparable with that obtained with the triple quadrupole. Barr says that they have already started doing some analyses on ion traps that were previously done on triple quadrupoles.
Analytical Chemistry News & Features, June 1, 1999
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