Electrospray ionization detection of inherently nonresponsive epoxides by peptide binding Nadja B. Cech, Jennifer R. Krone and Christie G. Enke* University of New Mexico, Department of Chemistry, 103 Clark Hall, Albuquerque, NM 87131, USA Received 2 January 2001; Revised 2 May 2001; Accepted 3 May 2001 A small organic molecule that is inherently nonresponsive to electrospray analysis, 1,3-butadiene diepoxide, was analyzed via electrospray ionization ESI) by binding it to various peptides and observing the product at the characteristic mass shift. The epoxide reacted only with peptides with arginines in their sequence, most likely through a base-catalyzed ring opening to form a covalently bound product. A calibration curve linear over 3 orders of magnitude was generated for the butadiene diepoxide/peptide adduct. Several other epoxides were also reacted with the peptide of choice angiotensin II), and adducts of these epoxides with the peptide were observed as well, demonstrating the versatility of this method for the analysis of small epoxides. This study demonstrates the possibility of assaying epoxides bound to peptides or proteins in biological samples. Furthermore, it demonstrates an important concept that could be applied to other analytical problems in electrospray: the ability to react an analyte that is nonresponsive to electrospray analysis with an analyte well suited for the technique, and accomplish quantitation based on the adduct formed between the two. Copyright # 2001 John Wiley & Sons, Ltd. For a compound to be well suited for analysis by electro- spray ionization mass spectrometry ESI-MS), several re- quirements must be met. Most importantly, the compound must be chargeable. Its ESI response will be improved by the presence of significant nonpolar portions, which increase its affinity for the ESI droplet surface. 1±4 Finally, the best ESI detection limits are usually accomplished with compounds of relatively high mass, since the low mass region in the mass spectrum is often complicated by background in the form of solvent clusters. ESI is therefore perfectly suited for the analysis of biomolecules, such as peptides, proteins and oligonucleotides, which fulfill all of the above requirements. On the other hand, small organic molecules are often difficult to analyze with ESI due to low proton affinity, low molecular weight and lack of nonpolar portions. Such is the case with epoxides. In this work, we show how inherently nonresponsive epoxides can be analyzed with ESI in the form of peptide adducts. The research presented here focuses on the analysis of 1,3- butadiene diepoxide BDO 2 ), a metabolite of butadiene. Butadiene is a chemical commonly used in the rubber industry, and thus workers in this industry are at risk of butadiene exposure. Butadiene has been shown to be carcinogenic in humans. 5±7 In studies of mice, rats and humans, it has been shown that this carcinogenic effect can be related to the metabolism of butadiene to form toxic epoxides, among them 1,3-butadiene diepoxide. 6,8 Further- more, 1,3-butadiene diepoxide itself is a common source of air pollution resulting from the emissions of automobiles, oil refineries and chemical plants. 9 Because of the toxicity of butadiene diepoxide, a rapid screening technique for this metabolite in plasma or urine is desirable. Currently, analysis of epoxide levels is accomplished with gas chroma- tography/mass spectrometry GC/MS), which requires lengthy vacuum distillation procedures to separate the epoxide from its nonvolatile plasma or urine matrices. 10 If butadiene diepoxide could be modified in such a way as to make its analysis with ESI-MS possible, this lengthy sample preparation could be eliminated. METHODS Stock solutions of peptides Bachem, Torrance, CA, USA) were made up at concentrations of 1.0  10 3 M. Each peptide/epoxide reaction was carried out in a 0.5 mM NaOH solution at 40 °C for 2 h. The peptide concentration in these solutions was 1  10 4 M, while the epoxide concentration varied according to the application see Results and Discussion section). The reaction solution was then diluted 10-fold into 50:50 methanol/water, 0.5% acetic acid solution, and analyzed using a TSQ-7000 mass spectro- meter Finnigan MAT, San Jose, CA, USA) with a modified 11 API source. A 50-mm i.d. fused silica capillary was used as the spray needle for all experiments, and was positioned at a distance of 1 mm from the entrance to the mass spectro- meter. A spray voltage of 4.5 kV was applied to the needle through direct electrical contact to a union 3 mm from the needle tip. A 5  10 10 O resistor was inserted in series with the ESI power supply in order to generate a stable ESI spray *Correspondence to : C. G. Enke, University of New Mexico, Department of Chemistry, 103 Clark Hall, Albuquerque, NM 87131, USA. E-mail: Enke@unm.edu Contract/grant sponsor: National Institutes of Health; Con- tract/grant number: GM44077. Contract/grant sponsor: P®zer, Inc. DOI:10.1002/rcm.337 Copyright # 2001 John Wiley & Sons, Ltd. RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2001; 15: 1040±1044