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.
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 characterization of proteins and peptides by mass spectrometry, including: (1) identification of proteins isolated by electrophoresis; (2) confirming molecular weights of recombinant or synthetic proteins and peptides; (3) determining sites of specific post-translational modifications including phosphorylation, glutamylation, AMPylation, and disulfide bond formation; (4) quantification of specific post-translational modifications; and (5) sequencing 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.
The facility currently has four mass spectrometers in use for specific areas of analysis.
ABI 4800 MALDI TOF/TOF
The state-of-the-art high-performance MALDI TOF (matrix-assisted laser desorption/ionization) TOF/(time-of-flight) instrument can be operated in both positive- and negative-ion modes and is 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 was delivered and installed in early 2014. It couples an 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 being used to determine collision cross-section measurements of ions for small molecules and intermolecular complexes and well as separation and analysis of complex mixtures of lipids and peptides.
Agilent 6460 LC-ESI QqQ (Triple Quad)
The instrument is currently being used for small-molecule analysis and quantification, principally for steroid profiling, and determination 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.
The Mass Spectrometry Facility currently serves between 10–15 sections within the institute, representing about 40 projects, as well as two PIs of sister institutes on the NIH campus and collaborations with five outside institutions.
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) for which reporter ion signals collected in the tandem MS experiment are used 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, the reporter ion ratios were found 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.
We developed a method to compare relative levels of specific proteins in the cerebrospinal fluid (CSF) from patients, using the 8-plex iTRAQ labeling process to mass-tag peptides generated from proteins present in each sample. The method will maintain individual patient information and provide the ability to compare results across multiple iTRAQ experiments. Initially, we used the Npc1 knock-out mouse model to determine the analytical variation of the method. The method is now being applied to 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. One family of proteins elevated in CSF from SLOS patients was the granin protein family. Examples include Chromogranin A, Secretogranin 3, Neurosecretory protein VGF, and ProSAAS. These secretory granule proteins are involved in the neuroendocrine secretory pathway and are stored and released with neuropeptides and hormones via a regulated exocytosis mechanism.
Gel-based proteomic studies in human genetic disorders
In collaboration with NICHD DIR clinical groups, several studies were performed by the facility to measure changes in protein expression in several animal models of human genetic disorders or in tissues from patients with the genetic disorders. Changes in protein expression were quantified by the intensity of spot staining for proteins separated by two-dimensional gel electrophoresis (2D-GE). Differentially expressed proteins were then identified by in-gel digestion and peptide analysis by MS/MS fragmentation. Studies using this approach were previously completed by this lab for two mouse models of human diseases: (1) Niemann-Pick Disease-Type C1 (NPC1); and (2) the tumor aggressiveness of pheochromocytomas/paragangliomas. Currently, we are using the approach to identify changes in protein expression in adrenal gland tissue from patients with a variety of adrenal-gland disorders, for which the genetic defect may not yet be determined.
Identification of amyloidosis-associated proteins in clinical samples
We developed a non-targeted approach to identify amyloidosis-associated proteins. The method was used to identify leukocyte cell–derived chemotaxin-2 (LECT2) as a component of amyloid plaques in adrenal tissue. Although the patient’s adrenal tissue was positive for amyloid by Congo Red staining, specific immunostaining for proteins commonly known to form amyloid plaques were all negative. In our non-targeted method, plaque proteins from disease tissue were extracted and separated by 1D SDS/PAGE. After digestion of the gel bands, we identified serum-amyloid P-component and LECT2 as components of the plaques. LECT2 has been reported in amyloid plaques in kidney tissue, but this is the first observation for this protein causing amyloidosis in adrenal tissue. The high accumulation of LECT2 in adrenal amyloid plaques from this patient was confirmed by Western blots using a specific anti-LECT2 antibody. The method demonstrates the advantage of using a non-targeted approach to detect novel proteins involved in amyloidosis.
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 at a much higher rate than is possible with liquid chromatography. In addition, IMS separations are associated with 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 to be undertaken 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 have been 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.
Quantitation of plasma melatonin (5-methoxy-N-acetyltryptamine) and N-acetyltryptamine
A multiple-reaction-monitoring (MRM)–based assay was developed in the Facility 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, rats, and rhesus monkeys. The liquid chromatography/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 that N-acetyltryptamine increases 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 chronobiotic though actions mediated by the melatonin receptor.
Mass spectrometry–based profiling of 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 owing to cross-reactivity with other steroids, and targeted LC-MS/MS is limited to the analysis of pre-determined analytes. We therefore developed a new LC-MS/MS approach to urinary steroid profiling that enables us to detect the steroids that have truly changed in a patient cohort without prior knowledge of the steroids' identity (i.e., untargeted metabolomics of steroids). Initially, the studies detected, in polycystic ovary syndrome (PCOS) patients, 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. The approach is being further extended to additional PCOS patients and to studies on patients with congenital adrenal hyperplasia (CAH). In a more targeted assay to quantitate specific steroids, we developed an MRM (multiple reaction monitoring) assay for quantitation of 5-α-pregnane-3α,17-α-diol-20-one (pdiol) and its 5-β stereoisomer, 17-α-hydroxypregnanolone (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, urinary levels of both pdiol and 5-β-pDiol were directly correlated with the serum levels of androstenedione. In addition, we have begun to develop a product ion spectrum database of known steroids to improve our ability to identify novel steroids.
The Mass Spectrometry 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.
- 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.
- Dolinska MB, Kovaleva E, Backlund P, Wingfield PT, Brooks, BP, Sergeev YV. Albinism causing mutations in recombinant human tyrosinase alter intrinsic enzymatic activity. PLoS One 2014; 9:e84494.
- Paul Blank, PhD, Program in Physical Biology, NICHD, Bethesda, MD
- Juan S. Bonifacino, PhD, Cell Biology and Metabolism Program, NICHD, Bethesda, MD
- William Clarke, PhD, The Johns Hopkins School of Medicine, Baltimore, MD
- Jens R. Coorssen, PhD, University of Western Sydney, Sydney, Australia
- Thomas E. Dever, PhD, Program in Cellular Regulation and Metabolism, NICHD, Bethesda, MD
- Peter Harrington, PhD, Ohio University, Athens, OH
- Stephen H. Leppla, PhD, Laboratory of Parasitic Diseases, NIAID, Bethesda, MD
- Matthias Machner, PhD, Cell Biology and Metabolism Program, 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, Program in Developmental Endocrinology and Genetics, NICHD, Bethesda, MD
- Dan Sackett, PhD, Program in Physical Biology, 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, Cell Biology and Metabolism Program, NICHD, Bethesda, MD
- Constantine A. Stratakis, MD, D(med)Sci, Program in Developmental Endocrinology and Genetics, NICHD, Bethesda, MD
- Joshua Zimmerberg, MD, PhD, Program in Physical Biology, NICHD, Bethesda, MD
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