Tandem mass spectrometry analysis of synthetic opioid peptide analogs.

Five synthetic opioid peptides that were designed to have specific opioid receptor-binding properties were studied by low energy collision-induced dissociation (CID) tandem mass spectrometry (MS/MS). The MS/MS data are required for the analysis of those peptides in ovine plasma in a study to determine the placental transfer of the peptide to the fetus. The synthetic enkephalin-related peptides were: Tyr-D-Arg-Phe-Lys-NH2, (DALDA), N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH, (ICI 174,864), Tyr-D-Thr-Gly-Phe-Leu-Thr, (DTLET), Tyr-D-Pen-Gly-Phe-D-Pen-OH, (DPDPE), and D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2, (CTAP). Liquid secondary ion mass spectrometry (LSIMS) was used for sample desorption-ionization, and a hybrid (E1BE2qQ) tandem mass spectrometer was used to collect the product-ion spectra. A protonated molecule ion, [M + H]+, was observed for each peptide. Amino acid sequence-determining fragment ion were produced by CID and collected by MS/MS for the three linear peptides, and also for the two disulfide-bond-containing peptides in their unreduced and dithiothreitol (DTT)-reduced forms. The detection level for the [M + H]+ ion of DTLET was ca. 3 pmol; and the stabilities of the CTAP and ICI analogs in plasma were studied.

Characterization of an opioid peptide-containing protein and of bovine α-lactalbumin by electrospray ionization and liquid secondary ion mass spectrometry.

Mass spectrometry methods have been used to characterize two proteins: an opioid peptide-containing protein extracted from bovine pituitary, and bovine α-lactalbumin (BAL). A protein that contains ß-endorphin was found in bovine pituitary, and that protein was characterized with electrospray ionization mass spectrometry (ESIMS), gel permeation chromatography, reversed-phase high performance liquid chromatography (RP-HPLC), radioimmunoassay, trypsinolysis, and liquid secondary ion mass spectrometry (LSIMS).BAL is a protein that was used as a model to develop analytical methods to study opioid peptide-containing proteins. Commercial BAL was purified by RP-HPLC, and its molecular weight (M.W.) was determined by ESIMS. The shift in mass observed following dithiothreitol (DTT) reduction estimated the number of disulfide bonds.For all of the data obtained for BAL with or without RP-HPLC separation, ESIMS determined the M.W. of the peptides produced by trypsin treatment of BAL, and LSIMS selected a precursor ion, the protonated molecule ion [M + H](+), of a tryptic peptide, which was analyzed by tandem mass spectrometry. Following DTT reduction, ESIMS and LSIMS detected each peptide that contained disulfide bonds in that mixture of tryptic peptides.

Methionine enkephalin-like, substance P-like, and beta-endorphin-like immunoreactivity in human parotid saliva.

These three neuropeptides were measured at daily baseline values by radioimmunoassay. Stimulated parotid saliva was collected from 31 subjects using a modified Carlson-Crittenden device affixed over Stenson's duct. Methionine enkephalin-like immunoreactivity ranged from 6.6 to 11.7 fmol/ml, with a mean of 9.3 fmol/ml. Substance P-like immunoreactivity ranged from 6.1 to 12.6 fmol/ml, with a mean of 9.3 fmol/ml. beta-Endorphin-like immunoreactivity ranged from 1.2 to 3.6 fmol/ml, with a mean of 2.6 fmol/ml. This is believed to be the first documentation of methionine enkephalin- and substance P-like activities in human parotid saliva and the first demonstration of beta-endorphin-like activity in any type of human saliva. Substance P-like activity was significantly higher in morning than evening samples; beta-endorphin-like activity also tended to be higher in the morning samples. Substance P and beta-endorphin-like immunoreactivities covaried in a significant positive manner, suggesting either common control mechanisms or similar responses to physiological variables.

Characterization of beta-endorphin in human pituitary by fast atom bombardment mass spectrometry of trypsin-generated fragments.

A novel mass spectrometric method possessing a high level of structural specificity is described for characterization in biological fluids and tissues of endogenous beta-endorphin of the human amino acid sequence (beta h-EP). The method is based upon purification of tissue extracts by an RP-HPLC gradient, followed by trypsinolysis of that particular HPLC fraction corresponding to the elution time of synthetic beta h-EP. The tryptic digest of that endogenous beta h-EP fraction was purified further by a second RP-HPLC gradient. A unique tryptic fragment selected from the second gradient was analyzed by fast atom bombardment mass spectrometry and B/E linked-field scan MS/MS techniques to provide molecular weight and amino acid sequence-determining fragment ion information, respectively, of that fragment. Collectively, these independent analytical methodologies provided unequivocal structure evidence for the presence of endogenous beta h-EP in human pituitary. The method was established first by utilizing synthetic beta h-EP to optimize experimental parameters, and then applied to the analysis of beta h-EP in post-mortem human pituitary extracts. The suitability of the present method for semi-quantitation of tissue extracts is also demonstrated. The corresponding detection limit of the synthetic beta h-EP was 90 fmol, and human pituitary contained 1.5 pmol of beta h-EP mg-1 protein. The method can be extended readily to the analysis of beta-endorphin derived from other species and tissues.

Effects of orthodontic force on methionine enkephalin and substance P concentrations in human pulpal tissue.

Orthodontic treatment typically involves intermittent periods of patient discomfort caused by forces on the teeth and adjacent tissues. This sensation of discomfort presumably is caused by the action of neuropeptides in the peripheral and central nervous systems. The effects of orthodontic force on the concentrations of two endogenous neuropeptides, methionine enkephalin (ME) and substance P (SP), measured as immunoreactive-methionine enkephalin (ir-ME) and immunoreactive-substance P (ir-SP), in human tooth pulp were evaluated in 20 patients from whom premolars were extracted before orthodontic treatment. The teeth from nine controls were not subjected to a force, whereas the 11 experimental patients had force applied to their maxillary premolars either by a transpalatal spring ligature or, in one case, by a headgear. The ligature applied a force within the range of 120 to 245 gm; the headgear applied 600 gm. Reversed-phase high-performance liquid chromatography (RP-HPLC) was used to purify the neuropeptides in the pulp homogenate, and radioimmunoassay (RIA) was used to quantify ir-ME and ir-SP in their appropriate HPLC fractions. (1) Females subjected to orthodontic force had significantly greater ir-ME concentrations than males. (2) The ir-SP concentration decreased significantly from the first to the third tooth extracted, then increased from the third to the fourth tooth. (3) Ir-SP and ir-ME concentrations are positively intercorrelated. The association was highest in the first tooth extracted from controls; surgical extraction decreased the correlation, although it continued to be positive. (4) The concentrations of ir-ME and ir-SP each correlated negatively with the magnitude of the orthodontic force and that correlation was enhanced when the value of the force was log-transformed.

Purification, characterization, and localization of neuropeptides in the cornea.

The immunologically detected neuropeptides methionine enkephalin (ME), substance P (SP), beta-endorphin (beta-End), and alpha-melanocyte stimulating hormone (alpha-MSH) were purified from bovine corneal extracts by gradient, followed by isocratic, reversed phase-high performance liquid chromatography (RP-HPLC) and characterized, after both chromatographic steps, by radioimmunoassay (RIA). Immunologically detected ME and SP were purified from canine corneal extracts by gradient RP-HPLC and characterized by RIA. An anatomical study of the bovine cornea separated the cornea into an epithelium-enriched and a stroma-enriched portion. After gradient RP-HPLC, RIA demonstrated that all the ME-like immunoreactivity was located in the corneal epithelium, whereas the SP-like immunoreactivity was distributed between the stroma and epithelium in an approximate two-to-one ratio.

Methionine-enkephalin peptides in human teeth.

The presence of the free opioid pentapeptide methionine enkephalin (ME) and of ME-containing peptide(s) was established firmly in decalcified, depulped human teeth by using a combination of methods including RP-HPLC, radioimmunoassay, radioreceptorassay, trypsin, carboxypeptidase B, fast atom bombardment mass spectrometry, and MS/MS methodology. Positive structural identification of ME was made with mass spectrometry. Those data demonstrate the presence of the preproenkephalinergic A system in the human trigeminal sensory termini.

Opioid peptide profile in human pituitary.

The comprehensive metabolic profile of endogenous opioid peptides is established here for human pituitary for the first time. Sixteen human pituitaries, obtained postmortem, were analyzed individually by gradient reversed-phase high-performance liquid chromatography together with a radio-receptor assay with [3H]etorphine as ligand. This combination was used to detect opioid receptor activity. The 16 assay profiles were sufficiently consistent for a composite of them to serve as a comparative basis for other studies on the pathophysiology of the human pituitary. To demonstrate one selected comparison, we present data on a distinctively different profile of opioid receptor activity in the pituitary of one patient who died from a drug overdose.

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

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 collected 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.

Profiling of neuropeptides using gradient reversed-phase high-performance liquid chromatography with novel detection methodologies.

Biological tissues and fluids are subjected to gradient reversed-phase high-performance liquid chromatography (RP-HPLC) separation and the neuropeptide profile of the collected fractions is obtained by radioreceptor assay (RRA) using a broad-based competing ligand. Radioimmunoassay (RIA) is also used to detect specific neuropeptides in the HPLC-purified fractions. Further confirmation of the identity of the peptides present in the tissue is obtained by mass spectrometry (MS) in the fast atom bombardment (FAB) mode. FAB-MS produces the protonated molecular ion of the peptide and allows direct measurement of underivatized peptides at the nanogram level, with increased molecular specificity. FAB-MS-MS identifies a unique amino acid sequence-determining ion in the mass spectrum of a peptide and offers maximum molecular specificity. This analytical chromatography methodology is applied to the study of the molecular basis of several disease states by monitoring several peptidergic pathways and individual peptides and their metabolic relationships. Molecular mechanisms involved in pain, stress, tumor formation, and neurological studies are studied.