A Mass Spectrometry Assay for Quantitation of N-Acetyltryptamine, N-Acetylserotonin and Melatonin (5-methoxy N-acetyltryptamine) in Plasma and Tissues. An MRM based assay to quantify N-acetyltryptamine and melatonin has been developed to study daily changes in tryptophan metabolites that may play a physiological role for actions mediated by the melatonin receptor. N-acetyltryptamine is known to be a mixed agonist/antagonist of the melatonin receptor. However, endogenous levels of this compound in mammals have not be previously observed. This assay has provided the first evidence for the presence of N-acetyltryptamine in plasma from human, rats, and rhesus monkeys (1). The liquid chromatography/tandem mass spectrometric method employs deuterated internal standards to quantitate these compounds. N-acetyltryptamine was detected in daytime plasma from human volunteers, rhesus macaques and rats. Twenty-four hour studies of rhesus macaque plasma revealed that N-acetyltryptamine increases at night to concentrations that exceed those of melatonin. These findings establish the physiological presence of N-acetyltryptamine in circulation and support the hypothesis that this compound may play a significant physiological role as an endocrine or paracrine chonobiotic though actions mediated by the melatonin receptor. Ion Mobility Mass Spectrometry for Detection of Ion Complexes. The Facility has the first commercial Ion Mobility Q-TOF LC/MS instrument (Agilent Technologies, Model 6560). The goal of implementing ion mobility spectrometry (IMS) prior to mass analysis is to add a dimension of separation to sample analysis that is orthogonal to both chromatography and mass spectrometry. Since the IMS operates on a millisecond time scale, the device offers the possibility of performing separations of complex mixtures at a much higher rate than possible with traditional LC methods. In addition, since IMS separations are associated with collision cross section (CCS) of ions (CCS is essentially a 'shape' parameter of ions in the gas phase), molecules of identical molecular weights can potentially be separated from one another on the basis of their CCS. This has implications for separations of isobaric steroids, lipids, peptides and proteins. IMS also offers the possibility of studying intermolecular complexes and their stoichiometry. One of the initial studies being undertaken is to investigate cyclodextrin-cholesterol complexes. We have also applied this technology to detect quantitative differences between different formulations of 2-hydroxypropyl-beta-cyclodextrin. These compounds have been used in a variety of therapeutic treatments and they are currently being tested for treatment of Niemann-Pick Disease Type C1 (NPC1). Formulations of this compound are complex mixtures with differing degrees of hydroxypropylation, and the degree of substitution is a critical aspect of the mixture, since these modifications can influence the binding to other molecules and potentially modulate the biological effects. Using IMS-MS analysis, we could demonstrate substantial differences in the degrees of substitution between two commercial products (2). Ion Mobility Mass Spectrometry for Improved Analysis of Phospholipids. The use of combined ion-mobility/mass spectrometry to analyze complex extracts of phospholipids has been investigated for the analysis of incorporation of branched chain fatty acids into phosphatidylcholine (PC) in mice fed a diet supplemented with the C20 branched chain alcohol, phytol. Phytol is metabolized to phytanic acid that is incorporated into phospholipids and triglycerides. Muscle tissue lipids were extracted and then analyzed by ion-mobility/mass spectrometry. To confirm the presence of phytanic acid, the ion mobility of these species was compared with diphytanoyl PC standard and endogenous straight chain PC phospholipids. The muscle phosphatidylcholine species profiles were similar between phytol and control diet, except that some additional species were detected in the phytol diet muscle. The two most abundant novel species (m/z 790.7 and 862.7) were tentatively identified as PC 20:0-16:0 and PC 20:0-22:6. To confirm the presence of phytanic acid in these PCs, the ion-mobility of these species were compared with diphytanoyl PC standard and endogenous straight chain PC phospholipids, and the ion-mobility of the novel PC species were consistent with incorporation of one branched chain phytanic acid. An Improved Method for Protein Identification that Incorporates Analysis of both MS and MS/MS Spectral Data. We recently developed a method for protein identifications based on the combined analysis of MALDI TOF MS and MS/MS spectral data collected from tryptic digests of proteins in gel bands. The method uses theoretical peptide masses and measurement errors observed in the matched MS spectra to confirm protein identifications obtained from a first pass MS/MS database search. The method makes use of the mass accuracy of the MS1-level spectral data that have heretofore been ignored by most peptide database search engines. A probability model was developed to analyze the distribution of mass errors of peptide matches in the MS1 spectrum to provide a confidence level to the additional peptide matches. These additional matches are independent of the MS/MS database search identifications, and provide additional corroboration to identifications from MS/MS-based scores that are otherwise considered to be only of moderate quality. This ProteinProcessor data analysis software that was developed is easily applicable to current proteomic analyses, provides a robust and invaluable addition to current protein identification tools (3). Mass Spectrometric-Based Analysis of Serum and Urinary Steroids. Current approaches to the analysis of urinary steroids typically employ either immunoassay or mass spectrometry based technologies. Immunoassay-based methods often lack specificity due to cross-reactivity with other steroids, while targeted LC-MS/MS is limited to the analysis of pre-determined analytes. We have developed a new LC-MS/MS approach to urinary steroid profiling that enables us to detect steroids that have truly changed in a patient cohort without knowing their identity beforehand (i.e., untargeted metabolomics of steroids). In addition, we have developed a product ion spectrum database of known steroids to improve our capability to identify novel steroids. We have also developed a reversed phase LC-MRM method to quantitate alpha and beta p-Diol levels in urine. These compounds are intermediates in the backdoor pathway for androgen synthesis. This assay has been applied to studies of patients with Congenital Adrenal Hyperplasia (CAH). Both alpha and beta p-Diol levels in urine were found to correlate with serum androgen levels, suggesting that these compounds could be novel biomarkers to monitor CAH disease control by glucocorticoid treatment. Current projects are developing an assay to quantitate glucocorticoids in mouse serum.
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