Measurement of opioid peptides with combinations of reversed phase high performance liquid chromatography, radioimmunoassay, radioreceptorassay, and mass spectrometry

Life Sciences, Vol. 41, pp. 809-812 Printed in the U.S.A.

Pergamon Journals

MEASUREMENT OF OPIOID PEPTIDES WITH COMBINATIONS OF REVERSED PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY, RADIOIMMUNOASSAY, RADIORECEPTORASSAY, AND MASS SPECTROMETRY Genevieve H. Fridland I and Dominic M. Desiderio 2,3 Charles B. Stout Neuroscience Mass^Spectrometry Laboratory I, and Departments of Neurology z and Biochemistry ~, University of Tennessee, Memphis 800 Madison Avenue, Memphis, Tennessee 38163

Summary Novel state-of-the-art mass spectrometric methods have been developed and are now used to identify and to quantify enkephalins and other neuropeptides in biological tissue extracts. As the first step, RP-HPLC gradient elution is performed of a Sep-Pak treated peptide-rich fraction from a tissue extract, and the eluent is monitored by a variety of post-HPLC detectors. In an effort to maximize the structural information that can be obtained from the analysis, UV (200 nm) provides the analog absorption trace; receptorassay analysis (RRA) data of all (90) fractions that are c o l l e ~ e d are used to construct the profile of opioid-receptoractive peptides; radioimmunoassay (RIA) of selected HPLC fractions at retention times corresponding to the retention time of standards, or in some special cases of all 90-fractions, provides immunoreactivity information; and fast atom bombardment mass spectrometry (FAB-MS) in two modes corroboration of the (M+H)+ of the expected peptide, or MS/MS to monitor an amino acid sequence-determining fragment ion unique to that peptide in the selected ion monitoring (SIM) mode provides structural information. As a demonstration of the level of quantification sensitivity that can be attained by these novel MS methods, FAB-MS-MS-SIM of solutions of synthetic leucine enkephalin was sensitive to the 70 femtomole level. This paper discusses RIA versus RRA data, and recent MS measurements of peptides in human tissues. The objective of this research is the identification and the accurate analytical measurement of endogenous opioid peptides that are extracted from biologic tissues, and to provide maximum molecular specificity and detection sensitivity with a combination of analytical techniques. We have developed a one0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.

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laboratory comprehensive analytical scheme that uses a variety of detection methods, each method based on the observation of a different molecular parameter of the peptide (i). Those post-HPLC detection methods include UV absorbance, receptor- and immunoreactivities, and two mass spectrometric methods: FAB-MS production of the protonated molecular ion to corroborate the indicated presence of a peptide, and MS/MS production of a unique amino acid sequence-determining fragment ion, which is used for quantification. At present, the greatest need in neurochemistry is not necessarily detection sensitivity but rather the crucial parameter molecular specificity. The latter datum is an experimental parameter that can be provided only by amino acid sequence information, which in turn can only be derived from gasphase, wet-chemical, or MS/MS sequencing methods. It is our opinion that mass spectrometric methods, while maximizing molecular specificity, can also attain a sensitivity that rivals that of the more generally used immunoassays. Methods Tissue preparation. Post-mortem human pituitaries are obtained and frozen within 12 hours of death. After acquisition, pituitary tumors, placental tissues, and human teeth are rapidly frozen in liquid nitrogen and kept at -70 ° C. The frozen tissue is weighed and homogenized in 1 M cold acetic acid, and an aliquot is taken for protein determination. Five volumes of acidified acetone (80% acetone; 20% .01M HCI) are added to further precipitate proteins. Following centrifugation, the supernate is dried, resuspended in 0.1% trifluoroacetic acid (TFA), and applied to a pre-wetted C18 Sep-Pak disposable cartridge; salts are removed by a TFA wash. The peptide-rich fraction is eluted with 50% acetonitrile in TFA, and lyophilized to dryness. HPLC separation. The peptide-rich fraction is loaded onto an octadecylsilyl analytical column; and a gradient elution separates the peptides. The nonlinear gradient is illustrated in Fig. i. UV absorbance is monitored at 200 nm; and one-minute fractions are collected. The triethylamine formic acid buffer is volatile to avoid interference with the post-HPLC detection of peptides. Radioreceptor assay (RRA). As an effective first step to determine those chromatographic fractions that contain opioid peptides, a radioreceptor assay is used to obtain a profile of opioid receptoractivity in the HPLC fractions (2). Tritiated etorphine, which is a broad-based ligand that competes with a wide range of opioid peptides is used as the competing ligand for binding to opioid receptors in a canine limbic system P2 preparation. Radioimmunoassay (RIA). Commercial RIA kits are used to identify and to quantify immunoreactivity (ir-) eluting from the gradient HPLC, especially in those fractions that contain opioid receptor binding activity. The immunoreactivity putatively due to ME, beta-endorphin, and other peptides of interest are measured, such as substance P (SP). The use of HPLC fractions for RIA minimizes but cannot ever avoid completely the danger of measuring closely-related co-eluting peptides that cross-react with the antibody.

Vol. 41, No. 7, 1987

Measurement of Opioid Peptides

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HPLC separation of the peptide-rich fraction from a pituitary chromophobe adenoma. The UV trace is shown on both chromatograms. RRA (top) and RIA (bottom) detection data are shown. The abscissa shows receptoractivity (top) and ME-like immunoreactivity (bottom); the ordinate shows fraction number; and the right axis percent acetonitrile (CH3CN). The gradient profile is shown as the connected straight lines from i0 to 30% CH3CN , followed by a 100% CH3CN column wash.

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Mass spectrometry (MS). Fast atom bombardment mass spectrometry is used to produce a (M+H)+ ion that provides the nominal mass of a peptide in those HPLC fractions that have demonstrated immuno- and/or receptoractivity. Mass spectrometry/spectrometry (MS/MS). In order to more accurately define the structure of the biological extract peptide to be measured, and to extend the information beyond the peptide's nominal mass, tandem mass spectrometry selects a unique amino acid sequence-determining fragment ion arising from that peptide. In principle, a first MS analysis produces the (M+H)+ ion, and a second MS produces the mass spectrum of that selected ion, which generally is the peptide (M+H)+ ion. In a two-sector MS, equivalent data are obtained with a B/E linked-field scan, which collects product ions arising from a precursor ion. Addition of known amounts of stable isotope-incorporated peptide internal standards, which possess identical HPLC and MS properties with the endogenous peptides but are collected at different masses, allow quantification of the endogenous peptide by determining the ratio of the integrated ion currents at both metastable reaction masses. Results and Discussion Opioid receptoractivity in a human chromophobe adenoma obtained from neurosurgery was profiled. All HPLC fractions were also assayed for ir-ME content using a commercial RIA kit (Fig. i). The fractions near the retention time of synthetic ME show immunoand opioid receptoractivities. An additional unidentified ME-like immunoreactive peak, which also demonstrate receptoractivity, was found in a more hydrophobic (later-eluting) area of the gradient elution. These data show the limited structural information and molecular specificity that can be provided by RIA. Furthermore, the two HPLC areas are still not guaranteed to be only one pure compound. In other studies, mass spectrometry in the FAB-MS/MS mode confirmed the presence of ME in the appropriate fractions from an HPLC-gradient from human teeth (submitted for publication). HPLC, RIA, RRA, and the MS-determined molecular weight of a peptide - when those analytical methods are used either individually or in combinations provide insufficient information concerning the structure of a peptide; only amino acid sequence-determining information provides structural certainty. MS/MS provides the highest level of molecular specificity to ensure identification of peptides in biological tissues (3), and recent FAB-MS/MS results indicate femtomole sensitivity (4). We plan to extend these studies to betaendorphin (submitted for publication), and C-terminally extended ME peptides. References i.

2. 3. 4.

G. FRIDLAND and D.M. DESIDERIO, J. Chromatogr. 379, 251-268 (1986). H. TAKESHITA, D.M. DESIDERIO, and G. FRIDLAND, Biomed. Chromatogr., l, 126-139 (1986). D.M. DESIDERIO, Analysis of Neuropeptides by Liquid Chromatography and Mass Spectrometry, Elsevier, Amsterdam, 1984. D.M. DESIDERIO and C. DASS, Analyt. Lett. 19, 1963-1971 (1986).