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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, 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. The Facility is located in the 9D corridor of Building 10 on the NIH campus.
Mode of operation
The Facility’s staff are available for consultation with all labs within the DIR, provided that existing resources are distributed equally among investigators requesting our 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. For each project, staff members meet with the Principal Investigator (PI) and other postdoctoral scientists involved in the study to discuss the experimental design. The Facility has an internationally recognized capability in characterization of proteins and peptides by mass spectrometry, including: (i) identification of proteins isolated by electrophoresis; (ii) confirming molecular weights of recombinant or synthetic proteins and peptides; (iii) determining sites of specific post-translational modifications including phosphorylation, glutamylation, AMPylation, and disulfide bond formation; (iv) quantification of particular post-translational modifications; and (v) 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 five 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–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 (IMS) with a high-resolution qTOF mass spectrometer. The 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 measurement 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 urinary steroid profiling and the determination of glycolytic pathway metabolites.
Thermo-Finnigan LCQ Deca:
The lower-resolution instrument is used exclusively for peptide characterization by nanoLC–electrospray ionization and fragmentation. It yields data that are complimentary to those produced by the MALDI TOF/TOF.
ABI Voyager MALDI TOF:
The instrument is used for the analysis of protein mixtures and to verify MWs of intact proteins. It is also available for general use after a prospective user has undergone appropriate training.
The Mass Spectrometry Facility currently serves 15 sections within the institute, representing about 40 projects, as well as two PIs of sister institutes on the NIH campus.
Proteomic studies in human genetic disorders
In collaboration with NICHD DIR clinical groups, several studies were performed by the facility to determine changes in protein expression in several animal models of human genetic disorders or in tissues from patients with the genetic disorders. In these studies, changes in protein expression were quantified by the intensity of spot staining for proteins separated by 2-dimentional 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 have been previously completed by this lab for two mouse models of human diseases: (i) Niemann-Pick Disease-Type C1 (NPC1), and (ii) tumor aggressiveness of pheochromocytomas/ paragangliomas. Currently, the approach is being applied 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 be determined.
Characterization of protein post-translational modifications
We characterized post-translational modification of Lys51 in recombinant eIF5A by measuring the molecular weight of the intact protein by LC/Q-TOF mass spectrometry (MS). The specific Lys is modified in a two-step enzymatic process to form hypusine. Deconvolution of the multiple charge states of the intact protein could detect both modification of Lys51 to deoxy-hypusine and hypusine. The effect of site-specific mutations in eIF5A on altering the level of Lys51 modification were also examined. In a separate project, N-linked glycosylation sites of recombinant human tyrosinase were characterized. For the recombinant protein, five of the potential seven Asn glycosylation sites were observed. From the sites, eight specific glycopeptides were observed and mapped to three specific Asn residues.
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 in 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, serum-amyloid P-component and LECT2 were identified as components of these 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.
Protein quantification in 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) where reporter ion signals collected in the tandem MS experiment are used for the 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. In our studies, the behavior of the iTRAQ 8-plex chemistry using MALDI-TOF/TOF instrumentation was evaluated. In order 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 replicated protein mixtures of equal concentration, the ion ratios were found to have a normal Gaussian distribution; the width of the distribution can be used to establish confidence levels for a given reporter ion ratio. We are now developing a method to compare relative levels of proteins in CSF from patients, using an 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, studies in the Npc1 knock-out mouse model were used to determine the analytical variation of the method. The method is now being applied to CSF from patients with Niemann-Pick Disease, type C1, in order to profile protein changes and to identify biochemical alterations correlated with disease progression and treatments.
Ion mobility mass spectrometry for detection of ion complexes
Earlier this year, the Facility accepted delivery and installation of the first commercial Agilent Technologies Model 6560 Ion Mobility Q-TOF LC/MS instrument. 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. Given that the IMS operates on a millisecond time scale, compared with the time scale of minutes for chromatography, the device offers the ability to perform separations of complex mixtures at a much higher rate than possible otherwise. 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 steroids, lipids, peptides, and proteins. IMS also offers the ability to study intermolecular complexes and determine their stoichiometry. One of the initial studies being undertaken is to investigate b-cyclodextrin–cholesterol complexes; preliminary results of these measurements suggest formation of a trimeric complex that incorporates calcium ions as a bridge.
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 quantitated 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-b stereoisomer, 17-α-hydroxypregnanolone (5-b-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-b-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 coordinator of the NIH Mass Spectrometry Interest Group.
- 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.
- 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. Endocr Pract 2014;in press.
- Crutchfield CA, Olson MT, Gourgari E, Nesterova M, Stratakis CA, Yergey AL. Comprehensive analysis of LC/MS data using pseudocolor plots. J Am Soc Mass Spectrom 2013;24:230-237.
- Olson MT, Breaud A, Harlan R, Emezienna N, Schools S, Yergey AL, Clarke W. Alternative calibration strategies for the clinical laboratory: application to nortriptyline therapeutic drug monitoring. Clin Chem 2013;59:920-927.
- 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
- 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, DMedSci, Program in Developmental Endocrinology and Genetics, NICHD, Bethesda, MD
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