NICHD Biomedical Mass Spectrometry Core Facility
- Peter S. Backlund, PhD, Staff Scientist, Acting Director
- Vince Pozsgay, PhD, Staff Scientist
- Nancy E. Vieira, MS, Senior Research Assistant
- 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 bio-molecules, 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 9D corridor of Building 10 on the NIH campus. The Mass Spectrometry Facility currently serves 9–12 laboratories within the NICHD intramural research program, representing about 30 projects, as well as two PIs of sister institutes on the NIH campus and collaborations with five outside institutions.
The Facility is committed to promoting mass-spectrometric aspects of proteomics and other mass-spectrometric analyses in NICHD’s DIR. We make serious efforts to educate investigators on the benefits and pitfalls of the techniques used in the Facility. In particular, we provide coaching on the principles of appropriate methods for sample isolation and staining of gels. 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 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 Principal Investigator (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) 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 6460 LC-ESI QqQ (Triple Quad)
The instrument 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
Improved method for protein identification by analysis of both MS and MS/MS spectral data
We developed a 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 1).
Protein quantification in tissues and cerebral spinal fluid (CSF) using isobaric tags
Protein quantification is an important aspect of proteome characterization and is crucial to understanding biological mechanisms and human diseases. Discovery-based or un-targeted studies have often used covalent tagging strategies (i.e., iTRAQ®, TMTTM), which use reporter ion signals collected in the tandem MS experiment for quantification. However, it has been difficult to establish the relative changes in reporter ion signals that are required to detect significant changes at the protein level. We studied the behavior of iTRAQ 8-plex chemistry using MALDI-TOF/TOF instrumentation. To better understand the behavior of the reporter ions, we evaluated the use of within-spectra normalization, which we termed “row-normalization.” When applied to replicate protein mixtures of equal concentration, we found the reporter ion ratios to have a normal Gaussian distribution around the expected ratio of 0.125. Therefore, the width of the distribution can be used to establish a confidence level for a given reporter ion ratio (Reference 3). The method is being applied to a study of CSF from patients with Smith-Lemli-Opitz syndrome (SLOS) or Niemann-Pick disease, type C1 (NPC-1), in order to profile protein changes and identify biochemical alterations correlated with disease progression and treatments.
Ion mobility mass spectrometry for detection of isobaric lipids and ion complexes
The Agilent Model 6560 Ion Mobility Q-TOF LC/MS instrument combines an ion mobility drift cell in front of a high-resolution Q-TOF mass spectrometer. Implementing ion mobility spectrometry (IMS) prior to mass analysis adds a dimension of separation to sample analysis that is orthogonal to both chromatography and mass spectrometry. Given that IMS operates on a millisecond time scale, the device offers the ability to perform 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 can be separated on the basis of their CCS, which has implications for separations of isobaric biomolecules, including numerous steroids, lipids, and peptides. IMS also offers the ability to study intermolecular complexes and determine their stoichiometry. One of the first studies we undertook was to investigate beta-cyclodextrin–cholesterol complexes; preliminary results of the measurements suggest formation of a trimeric complex that incorporates calcium ions as a bridge. In addition, pH–dependent changes in cyclodextrin conformations were suggested by the observation of two distinct drift times of isobaric species and by an indication of changes in CCS attributable to two distinct conformations of the ions. We have begun molecular modeling studies of the cyclodextrin molecules to explain the two conformational states.
We used combined ion-mobility/mass spectrometry to analyze complex extracts of phospholipids to analyze branched-chain fatty acid incorporation into phosphatidylcholine (PC) in mice 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 phosphatidylcholine species profiles of 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 (m/z 790.7 and 862.7) as PC 20:0-16:0 and PC 20:0-22:6. To confirm the presence of phytanic acid in these PC’s, we compared the ion mobility of these species with a diphytanoyl PC standard and endogenous straight-chain PC phospholipids; the ion mobility of the novel PC species was consistent with incorporation of one branched chain phytanic acid.
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 the presence of N-acetyltryptamine in plasma from human, rat, and rhesus monkey. The LC/tandem mass spectrometric method employs deuterated internal standards to quantitate N-acetyltryptamine and melatonin. N-acetyltryptamine was detected in daytime plasma from human volunteers, rhesus macaques, and rats. Twenty-four-hour studies of rhesus macaque plasma revealed increases in N-acetyltryptamine at night to concentrations that exceed those of melatonin. 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.
Mass spectrometry–based profiling of urinary steroids
We developed a novel un-targeted LC-MS/MS approach to profile urinary steroids, which permits detection of steroids that have truly changed in a patient cohort without prior knowledge of the steroids’ identity (i.e., untargeted metabolomics of steroids). Initial studies in polycystic ovary syndrome (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. We are extending the approach to additional PCOS patients and to studies on patients with congenital adrenal hyperplasia (CAH). We also developed a targeted assay to quantitate specific urinary steroids, using an MRM (multiple reaction monitoring) assay for quantification of 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. Using this assay 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 2). In addition, we began to develop a product-ion spectrum database of known steroids to improve our ability to identify novel urinary steroids.
Identification of LECT2-associated amyloidosis in adrenal tissue
We recently identified leukocyte cell–derived chemotaxin-2 (LECT2) as a component in the formation of amyloid plaques in adrenal tissue (Reference 5). Although the adrenal tissue from a patient was positive for amyloid by Congo Red staining, specific immuno-staining for proteins commonly known to form amyloid plaques were all negative. We extracted plaque proteins from disease tissue and separated them by 1D SDS/PAGE. After digestion of the gel bands, we identified serum amyloid P-component and leukocyte cell–derived chemotaxin-2 (LECT2) in the plaques (Reference 5). Other reports had previously observed LECT2 in amyloid plaques in kidney, but this is the first observation of LECT2 amyloidosis in adrenal tissue. We confirmed the high accumulation of LECT2 in adrenal amyloid plaques from this patient by Western blots (using a LECT2–specific antibody), which were positive for the disease tissue but negative for comparable amounts of tissue from normal adrenal glands.
Publications
- 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.
- 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.
- Cologna SM, Crutchfield CA, Searle BC, Blank PS, Toth CL, Ely AM, Picache JA, Backlund PS, Wassif CA, Porter FD, Yergey AL. An efficient approach to evaluate reporter ion behavior from MALDI-MS/MS data for quantification studies using isobaric tags. J Proteome Res 2015;14:4169-4178.
- Pu J, Schindler C, Jia R, Jarnik M, Backlund P, Bonifacino JS. BORC, a multisubunit complex that regulates lysosome positioning. Dev Cell 2015;33:176-188.
- Rauschecker ML, Cologna SM, Xekouki P, Nilubol N, Shamburek RD, Merino M, Backlund PS, Yergey AL, Kebebew E, Balow JE, Stratakis CA, Abraham SB. Clinical Case Report: LECT2-associated adrenal amyloidosis. AACE Clinical Case Reports 2015;1:e59-e67.
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
- Jens R. Coorssen, PhD, University of Western Sydney, Sydney, Australia
- Thomas E. Dever, PhD, Section on Protein Biosynthesis, NICHD, Bethesda, MD
- 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, Bone and Extracellular Matrix Branch, 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
- Yuri V. Sergeev, PhD, Ophthalmic Genetics and Visual Function Branch, NEI, Bethesda, MD
- 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 Cellular and Membrane Biophysics, NICHD, Bethesda, MD
Contact
For more information, email backlunp@mail.nih.gov.
