National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2017 Annual Report of the Division of Intramural Research

NICHD Biomedical Mass Spectrometry Core Facility

Peter Backlund
  • Peter S. Backlund, PhD, Staff Scientist, Acting Director
  • Vince Pozsgay, PhD, Staff Scientist
  • Alfred L. Yergey, PhD, Scientist Emeritus

The NICHD Biomedical Mass Spectrometry Core Facility was created under the auspices of the Office of the Scientific Director to provide high-end mass-spectrometric services to scientists within the NICHD Division of Intramural Research (DIR). Particular focus has been in the areas of proteomics, biomarker discovery, protein characterization, and detection of post-translational modifications. The Facility also performs quantitative analyses of small biomolecules, including lipids and steroids. In addition, the Facility develops and modifies methods for the isolation and detection of biomolecules by mass spectrometry, as well as novel methods for data analysis. The Facility is located in the 8S corridor of Building 10 on the NIH campus. The Mass Spectrometry Facility currently serves both clinical and basic research laboratories within the NICHD intramural research program. As resources permit, we also collaborate with principal investigators (PIs) of other institutes within NIH and with other outside institutions.

The Facility is committed to promoting mass-spectrometric aspects of proteomics and other mass-spectrometric analyses in NICHD’s DIR. We make substantial efforts to educate investigators on the benefits and pitfalls of the techniques used in the Facility. In particular, we provide advice and protocols for appropriate methods of sample isolation that are compatible with analysis by mass spectrometry. We also support an NIH–wide seminar series featuring internationally known experts in proteomics. In parallel, the staff of the Facility have developed collaborations with other Institutes to promote exchange of information and to bring new mass-spectrometric techniques to the NICHD. In addition, Peter Backlund is the moderator of the NIH Mass Spectrometry Interest Group.

Mode of operation

The Facility is available to all labs within the DIR, provided that existing resources are distributed equally among investigators requesting services. The philosophy of the Facility is to ensure that its instruments obtain only reliable, high-quality data and that its clients receive only statistically meaningful analyses. The Facility’s staff are available for consultation on both project design and data interpretation. Before the start of a project, staff members meet with the PI and other scientists involved in each study to discuss experimental goals and data requirements. The Facility has an internationally recognized capability in the characterization of proteins and peptides by mass spectrometry, including: (1) identification of proteins isolated by electrophoresis; (2) confirmation of molecular weights of recombinant or synthetic proteins and peptides; (3) determination of sites of specific post-translational modifications, including phosphorylation, glutamylation, AMPylation, and disulfide bond formation; (4) quantification of specific post-translational modifications; and (5) sequencing of peptides de novo. In addition, the Facility has extensive experience and skill in the identification and quantification of small endogenous molecules including phospholipids, steroids, and sugars. In this latter area, the capability is primarily in quantification of endogenous levels of particular molecules and their metabolites.

Instrumentation

The facility currently has four mass spectrometers in use for specific areas of analysis.

SimulTOF 300 MALDI TOF/TOF

The state-of-the-art high-performance MALDI (matrix-assisted laser desorption/ionization) TOF/TOF (time-of-flight/time-of-flight) instrument can be operated in either positive- or negative-ion modes. The instrument is most often used for peptide identification in peptide mixtures without chromatographic separation. Methodology is also available to perform off-line liquid chromatography (LC) separation and sample spotting. Additional uses include relative peptide quantification for iTRAQ (isobaric tags for relative and absolute quantitation)–labeled peptides and sequence determination through de novo sequencing techniques for unusual peptides not present in gene-based protein databases.

Agilent 6560 Ion Mobility-qTOF

The state-of-the art instrument couples a one-meter ion-mobility-drift cell with a high-resolution qTOF mass spectrometer. Ion-mobility spectrometry (IMS) prior to mass analysis provides an added dimension of sample separation that is orthogonal to both chromatography and mass spectrometry. The instrument is currently used to determine collision cross-section measurements of ions for small molecules and intermolecular complexes and for separation and analysis of complex mixtures of lipids and peptides.

Agilent 6495 LC-ESI QqQ (Triple Quad)

The instrument is coupled to an Infinity 1290 UPLC system with either an ESI or APCI ion source, and is currently used for small-molecule analysis and quantification, principally for steroid profiling and the analysis of amino acid and glycolytic pathway metabolites.

ABI Voyager MALDI TOF

The instrument is used for the analysis of protein mixtures and to verify molecular weights of intact proteins. It is also available for general use after a prospective user has undergone appropriate training.

Major projects

Ion-mobility mass spectrometry for detection of isobaric biomolecules and ion complexes

Given that ion-mobility spectrometry (IMS) operates on a millisecond time scale, the technique performs separations of complex mixtures much faster than is possible with liquid chromatography (LC). In addition, IMS separations are associated with the collision cross section (CCS) of ions (CCS is essentially a ‘shape’ parameter of ions in the gas phase), so that molecules of identical molecular weights but with different structures can be separated on the basis of their CCS. This has great potential for separating isobaric biomolecules, including numerous steroids, lipids, and peptides. In addition to the separation of structural isomers, IMS also offers the ability to study intermolecular complexes in the gas phase to determine conformational changes and stoichiometry. One of the first studies we undertook with the Core Facility’s Agilent Model 6560 Ion Mobility Q-TOF LC/MS instrument was to investigate beta-cyclodextrin–cholesterol complexes in the presence of different monovalent and divalent cations. The measured CCS of different ions could be attributed to two distinct conformations of the ions, and we have begun molecular modeling studies to independently explain the different conformational states. More recently, this instrument has been used to detect structural differences between two commercial preparations of hydroxypropyl-modified beta-cyclodextrins (Reference 1). One of these preparations is currently being used in clinical trials to treat Neimann-Pick disease type C1 patients.

We are currently using ion-mobility/mass spectrometry to analyze complex mixtures of phospholipids extracted from mouse tissues in order to analyze branched-chain fatty acid incorporation into phosphatidylcholine (PC) in animals fed a diet supplemented with phytol, a saturated C20 branched-chain alcohol. Phytol is metabolized to phytanic acid, which can be incorporated into phospholipids and triglycerides. Muscle-tissue lipids were extracted and then analyzed by ion-mobility/mass spectrometry. The muscle PC species profiles under the phytol and control diet were similar, except that some additional species were detected in the phytol-diet muscle. We tentatively identified the two most abundant novel species as PC 20:0-16:0 and PC 20:0-22:6. The drift times for these novel species are consistent with the molecules containing the branched phytanoyl fatty acyl group. The separation of phospholipids by ion mobility also makes it possible to quantitate complex mixtures of PC species without the longer time period required for LC separation of these mixtures, shortening run times from 60 to 3 minutes of IMS separation.

Quantitation of plasma melatonin (5-methoxy-N-acetyltryptamine) and N-acetyltryptamine

We developed a multiple reaction monitoring (MRM)–based assay to quantify N-acetyltryptamine and melatonin in plasma. N-acetyltryptamine is a melatonin-receptor mixed agonist/antagonist. The assay provided the first evidence for endogenous N-acetyltryptamine in the daytime plasma from human volunteers, rhesus monkey, and rats. The mass-spectrometric method employs deuterated internal standards to quantitate N-acetyltryptamine and melatonin. Twenty-four-hour studies of rhesus macaque plasma revealed increases in N-acetyltryptamine at night to concentrations that exceed those of melatonin. We also used the technique to measure these compounds in tissues known to be involved in melatonin biosynthesis, and N-acetyltryptamine was present in both pineal and retinal tissue from rhesus macaques. The findings establish the physiological presence of N-acetyltryptamine in the circulation and support the hypothesis that this tryptophan metabolite plays a significant physiological role as an endocrine or paracrine chrono-biotic though actions mediated by the melatonin receptor (Reference 2).

Mass spectrometry–based profiling and quantification of serum and urinary steroids

We previously developed an MRM–based mass-spectrometry method to quantify several androgenic steroids in urine and applied the method to studies of polycystic ovary syndrome (PCOS) patients and patients with congenital adrenal hyperplasia (CAH). The assay was used to quantify 5-alpha-pregnane-3-alpha,17-alpha-diol-20-one (known also as pdiol) and its 5-beta stereoisomer, 17-alpha-hydroxypregnanolone (known also as 5-β-pdiol); pdiol is an intermediate in the ‘backdoor pathway’ from 17OH progesterone to dihydrotestosterone. In a study of CAH patients, we found urinary levels of both pdiol and 5-β-pdiol to be directly correlated with the serum levels of androstenedione (Reference 3). The assay also measures etiocholanolone, androsterone, and testosterone. More recently, we also developed an MRM–based assay for quantification of glucocorticoids in serum, including cortisol, cortisone, 11-deoxycortisol, and corticosterone.

In addition to the targeted assays, we also developed a novel un-targeted LC-MS/MS approach to profile urinary steroids, which permits detection of steroids that have changed in a patient cohort without prior knowledge of the steroids’ identity (i.e., untargeted metabolomics of steroids). Initial studies in PCOS patients detected elevated levels of an unknown compound consistent with an androgenic steroid. We were then able to identify the unknown as a mixture of androsterone-sulfate and etiocholanolone-sulfate. In order to expand this approach, it would be helpful to compare observed unknown peaks with a database of known steroid standards, and we are currently developing a spectral database that would be compatible with this type of spectral search.

Improved method for protein identification by analysis of both MS and MS/MS spectral data

We developed an improved method for protein identifications based on the combined analysis of MS and MS/MS spectral data collected from tryptic digests of proteins in gel bands using MALDI TOF-TOF instrumentation. The method uses theoretical peptide masses and the 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 heretofore were ignored by most peptide database search engines. We developed a probability model to analyze the distribution of mass errors of peptide matches in the MS1 spectrum and to thus provide a confidence level to the additional peptide matches. The 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 of only moderate quality. Straightforward and easily applicable to current proteomic analyses, this ‘ProteinProcessor’ provides a robust and invaluable addition to current protein identification tools (Reference 4).

Publications

  1. Yergey AL, Blank PS, Cologna SM, Backlund PS, Porter FD, Darling AJ. Characterization of hydroxypropyl-beta-cyclodextrins used in the treatment of Niemann-Pick Disease type C1. PLoS One 2017;12:e0175478.
  2. Backlund PS, Urbanski HF, Doll MA, Hein DW, Bozinoski M, Mason CE, Coon SL, Klein DC. Daily rhythm in plasma N-acetyltryptamine. J Biol Rhythms 2017;32:195-211.
  3. Debono M, Mallappa A, Gounden V, Nella AA, Harrison RF, Crutchfield CA, Backlund PS, Soldin SJ, Ross RJ, Merke DP. Hormonal circadian rhythms in patients with congenital adrenal hyperplasia: identifying optimal monitoring times and novel disease biomarkers. Eur J Endocrinol 2015;173:727-737.
  4. Epstein JA, Blank PS, Searle BC, Catlin AD, Cologna SM, Olson MT, Backlund PS, Coorssen JR, Yergey AL. ProteinProcessor: probabilistic analysis using mass accuracy and the MS spectrum. Proteomics 2016;16:2480-2490.

Collaborators

  • Paul Blank, PhD, Section on Cellular and Membrane Biophysics, NICHD, Bethesda, MD
  • Juan S. Bonifacino, PhD, Section on Intracellular Protein Trafficking, NICHD, Bethesda, MD
  • Stephanie M. Cologna, PhD, University of Illinois, Chicago, IL
  • Jens R. Coorssen, PhD, Brock University, St. Catharines, Ontario, Canada
  • Peter Harrington, PhD, Ohio University, Athens, OH
  • David C. Klein, PhD, Section on Neuroendocrinology, NICHD, Bethesda, MD
  • Stephen H. Leppla, PhD, Laboratory of Parasitic Diseases, NIAID, Bethesda, MD
  • Matthias Machner, PhD, Section on Microbial Pathogenesis, NICHD, Bethesda, MD
  • Joan Marini, MD, PhD, Section on Heritable Disorders of Bone and Extracellular Matrix, NICHD, Bethesda, MD
  • Deborah P. Merke, MD, MS, Pediatric Consult Service, NIH Clinical Center, Bethesda, MD
  • Matthew Olson, MD, The Johns Hopkins University Medical School, Baltimore, MD
  • Forbes Porter, MD, PhD, Section on Molecular Dysmorphology, NICHD, Bethesda, MD
  • Dan Sackett, PhD, Section on Cell Biophysics, NICHD, Bethesda, MD
  • Brian Searle, Proteome Software, Inc., Portland, OR
  • Stephen E. Stein, PhD, National Institute of Standards and Technology, Gaithersburg, MD
  • Gisela Storz, PhD, Section on Environmental Gene Regulation, NICHD, Bethesda, MD
  • Constantine A. Stratakis, MD, D(med)Sci, Section on Endocrinology and Genetics, NICHD, Bethesda, MD
  • Joshua Zimmerberg, MD, PhD, Section on Integrative Biophysics, NICHD, Bethesda, MD

Contact

For more information, email backlunp@mail.nih.gov.

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