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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