Ion Mobility Mass Spectrometry for Detection of Ion Complexes. 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 degree of substitution between two commercial products (3). Ion Mobility Mass Spectrometry for Improved Analysis of Phospholipids. The use of combined ion-mobility/mass spectrometry to analyze complex mixtures of phospholipids has been applied to the analysis of branched chain fatty acid incorporation into phosphatidylcholine (PC). To confirm the presence of the branched chain C20 phosphocholine 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 were tentatively identified as PC 20:0-16:0 and PC 20:0-22:6. The formation of PC complexes has also been demonstrated using a mixture of two purified PCs, and clusters of doubly-charged ions ranging from trimers to octamers were observed. The CCS values of the hepta- and octamermeric clusters show a significant departure from the trend line exhibited for trimers through hexamers, indicating a change in complex structure that may represent a transition to lipid self-organization. Assay for Quantitation of Phosphatidyl-Inositol mono-, di- and tri-phosphates. An MRM assay method was implemented to measure levels of phosphatidyl inositol phosphates, and this assay has been used to quantitate levels of these compounds in brain tissues and influenza virus particles. 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 Protein Processor data analysis software that was developed is easily applicable to current proteomic analyses, provides a robust and invaluable addition to current protein identification tools. Mass Spectrometric-Based Analysis of Serum and Urinary Steroids. We previously developed an LC-MS/MS approach to urinary steroid profiling that enabled detection of steroids that were 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 also developed a reversed phase LC-MRM method to quantitate alpha and beta p-Diol levels in urine. This assay has been applied to studies of patients with Congenital Adrenal Hyperplasia (CAH) and the results suggested that these compounds could be novel biomarkers to monitor CAH disease control by glucocorticoid treatment. In a current project, an assay was developed to quantitate a panel of glucocorticoids in mouse serum, including cortisol, cortisone, corticosterone, and 11-deoxycortisol. 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 (2). N-acetyltryptamine was detected in daytime plasma from human volunteers, rhesus macaques and rats, and twenty-four hour study 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 chronobiotic though actions mediated by the melatonin receptor.

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21
Fiscal Year
2018
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U.S. National Inst/Child Hlth/Human Dev
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Klein, Christian; Cologna, Stephanie M; Kurulugama, Ruwan T et al. (2018) Cyclodextrin and malto-dextrose collision cross sections determined in a drift tube ion mobility mass spectrometer using nitrogen bath gas. Analyst 143:4147-4154
Backlund, Peter S; Urbanski, Henryk F; Doll, Mark A et al. (2017) Daily Rhythm in Plasma N-acetyltryptamine. J Biol Rhythms 32:195-211
Yergey, Alfred L; Blank, Paul S; Cologna, Stephanie M et al. (2017) Characterization of hydroxypropyl-beta-cyclodextrins used in the treatment of Niemann-Pick Disease type C1. PLoS One 12:e0175478
Epstein, Jonathan A; Blank, Paul S; Searle, Brian C et al. (2016) ProteinProcessor: A probabilistic analysis using mass accuracy and the MS spectrum. Proteomics 16:2480-90
Pu, Jing; Schindler, Christina; Jia, Rui et al. (2015) BORC, a multisubunit complex that regulates lysosome positioning. Dev Cell 33:176-88
Debono, Miguel; Mallappa, Ashwini; Gounden, Verena et al. (2015) Hormonal circadian rhythms in patients with congenital adrenal hyperplasia: identifying optimal monitoring times and novel disease biomarkers. Eur J Endocrinol 173:727-37
Cologna, Stephanie M; Crutchfield, Christopher A; Searle, Brian C et al. (2015) An Efficient Approach to Evaluate Reporter Ion Behavior from MALDI-MS/MS Data for Quantification Studies Using Isobaric Tags. J Proteome Res 14:4169-78
Dolinska, Monika B; Kovaleva, Elena; Backlund, Peter et al. (2014) Albinism-causing mutations in recombinant human tyrosinase alter intrinsic enzymatic activity. PLoS One 9:e84494
Crutchfield, Christopher A; Olson, Matthew T; Gourgari, Evgenia et al. (2013) Comprehensive analysis of LC/MS data using pseudocolor plots. J Am Soc Mass Spectrom 24:230-7
Olson, Matthew T; Breaud, Autumn; Harlan, Robert et al. (2013) Alternative calibration strategies for the clinical laboratory: application to nortriptyline therapeutic drug monitoring. Clin Chem 59:920-7

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