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NICHD Biomedical Mass Spectrometry Facility

Peter S. Backlund, PhD
  • Peter S. Backlund, PhD, Staff Scientist, Acting Director
  • Vince Pozsgay, PhD, Staff Scientist
  • Christopher A. Crutchfield, PhD, Intramural Research Training Award Fellow
  • Nancy E. Vieira, MS, Senior Research Assistant
  • Stephanie Cologna, PhD, Intramural Research Training Award Fellow
  • Elizabeth Kahuno, BS, Contractor
  • 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 Intramural Research Program (IRP). 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 IRP, 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 internationally recognized capability in characterization of proteins and peptides by mass spectrometry including: (i) identification of proteins isolated by electrophoretic gels; (ii) confirming molecular weights of recombinant or synthetic proteins and peptides; (iii) determining sites of specific post-translational modifications including phosphorylation, glutamylation, AMPylation, and disulfides; (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 focused primarily on quantification of endogenous levels of particular molecules and their metabolites.

Instrumentation

The facility currently has five mass spectrometers (MS) in use for specific areas of analysis.

  1. 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.
  2. Agilent 6520 LC-ESI qTOF: The hybrid instrument produces high-resolution MS and MS/MS spectra for a variety of discovery projects. Current applications include the analysis of serum cardiolipins, protein molecular weight (MW) characterization, and peptide identification.
  3. 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.
  4. 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.
  5. 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 potential user has undergone appropriate training.

Facility usage

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.

Major projects

Proteomic studies in human genetic disorders:

The facility has performed many studies in collaboration with NICHD IRP clinical groups to determine changes in protein expression in several animal models of human genetic disorders or for tissues from patients with these genetic disorders. In such studies, 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 recently completed for two mouse models of human diseases: (i) Niemann-Pick Disease-Type C1 (NPC1), and (ii) tumor aggressiveness of pheochromocytomas/paragangliomas. For the NPC1 mouse model, Npc1 knock-out and wild-type mice were compared, and 77 differentially expressed proteins were identified from mouse cerebellum. Translating these findings from the animal model to cerebrospinal fluid (CSF) from NPC1 patients, three proteins with altered expression in the animal model (glutathione-S-transferase a, superoxide dismutase, and FABP3) were also found to have altered expression in CSF from NPC patients. 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, where the genetic defect may not be determined.

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. Although adrenal tissue was positive for amyloid by Congo Red staining, specific immunostaining for proteins commonly known to form amyloid plaques were all negative. Plaque proteins from disease tissue were extracted and separated by 1-D 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 the patient was confirmed by Western blots using a specific LECT2 antibody, which were positive for the disease tissue, while negative for comparable amounts of tissue from normal adrenal glands.

Protein profiling and quantification in CSF:

Two complementary approaches were applied to characterize protein biomarkers in CSF from patients with NPC. Significant differences between proteins in CSF from NPC patients and adult controls were demonstrated using a novel analytical approach that we developed. In this approach, we analyzed MALDI spectra by combining ANalysis Of Variance with Principal Component Analysis (ANOVA-PCA). We are now developing a method to compare relative levels of specific 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 permit comparison of results across multiple iTRAQ experiments. Initially, studies in the Npc1 knock-out mouse model are being used to determine the analytical variation of this method. Several proteins identified in our pilot study were also identified by 2D-GE in our previous study of differentially expressed cerebellar proteins. One example of a differentially expressed protein detected by both methods was ATP-synthase alpha, which showed a 2.6-fold decrease by 2D-GE while initial iTRAQ experiments resulted in 1.7-fold decrease. An additional protein that was found only in the iTRAQ experiment was Purkinje Cell Protein 4, which was down-regulated in the mutant mouse 1.9-fold. Purkinje cells are known to be the first neuronal cell type to succumb in NPC1 disease. The method is now being applied to CSF from patients with NPC1 in order to profile protein changes and identify biochemical alterations correlated to disease progression and treatments.

Lipid characterization and quantification in serum:

We developed a methodology to extract and quantify cardiolipins (CLs) in human serum. CLs are a major lipid in mitochondrial membranes. However, CLs can also be detected in serum, and their metabolism in serum has not been previously characterized. The analytical approach involves addition of an internal standard to a serum sample, followed by liquid-liquid and solid phase extractions. Cardiolipins are then detected and quantified by LC-MS analysis. Using this method, we analyzed cardiolipins in normal adult serum, plasma, and whole blood. The major CL in serum was identified as (18:2)-4 cardiolipin, in a concentration range between 3–10 nM, approximately 1000-fold lower than reported by earlier, less accurate measurements. In addition, cardiolipins with other fatty acid moieties were detected in serum using this approach, and the fatty acid composition of serum CLs was distinct from the composition found in plasma or whole blood. The method is currently being applied to quantify cardiolipins in maternal serum, cord blood, and infant serum from a clinical study. A novel family of CLs was identified in some of these patient samples and appears to be associated with bacterial infections and antibiotic treatment.

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 developed a new LC-MS/MS approach to urinary steroid profiling that enables us to detect, without priot knowledge of their identity, the steroids that have truly changed in a patient cohort (i.e., untargeted metabolomics of steroids). In addition, we utilized a product ion spectrum database of known steroids to improve our ability to identify novel steroids. The methods were tested in a pilot project to investigate urinary steroid for patients diagnosed with polycystic ovarian syndrome (PCOS). Initially, the studies detected elevated levels of an unknown compound consistent with an androgenic steroid in PCOS patients. We were then able to identify the unknown as a mixture of androsterone-sulfate and etiocholanolone-sulfate. Two-dimensional LC, using C18 in the first dimension and Porous Graphitic Carbon in the second, was required to separate the diastereomeric pair. The approach is being further extended to additional PCOS patients and to studies on patients with congenital adrenal hyperplasia (CAH).

Community outreach

The Mass Spectrometry Facility is committed to promoting mass-spectrometric aspects of proteomics and other mass-spectrometric analyses in NICHD's Division of Intramural Research. 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.

Publications

  1. Fliedner SM, Kaludercic N, Jiang XS, Hansikova H, Hajkova Z, Sladkova J, Limpuangthip A, Backlund PS, Wesley R, Martiniova L, Jochmanova I, Lendvai NK, Breza J, Yergey AL, Paolocci N, Tischler AS, Zeman J, Porter FD, Lehnert H, Pacak K. Warburg effect’s manifestation in aggressive pheochrocytomas and paragangliomas: insights from a mouse cell model applied to human tumor tissue. PLoS One 2012;7:e40949.
  2. Cologna SM, Jiang XS, Backlund PS, Cluzeau CV, Dail MK, Yanjanin NM, Siebel S, Toth CL, Jun HS, Wassif CA, Yergey AL, Porter FD. Quantitative proteomic analysis of Niemann-Pick disease, type C1 cerebellum identifies protein biomarkers and provides pathological insight. PLoS One 2012;7:e47845.
  3. 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.
  4. 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.

Collaborators

  • Paul Blank, PhD, Program in Physical Biology, NICHD, Bethesda, MD
  • William Clarke, PhD, The Johns Hopkins School of Medicine, Baltimore, MD
  • Jens R. Coorssen, PhD, University of Western Sydney, Australia
  • Jonathan Epstein, MS, Molecular Genomics Laboratory Core Facility, NICHD, Bethesda, MD
  • Peter Harrington, PhD, Ohio University, Athens, OH
  • Rachel O. Loo, PhD, Molecular Biology Institute, UCLA, Los Angeles, CA
  • Mattias Machner, PhD, Cell Biology and Metabolism Program, NICHD, Bethesda, MD
  • Deborah P. Merke, MD, 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
  • John Robbins, MD, Program in Developmental and Molecular Immunity, 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 Clinical Genetics Section, NEI
  • Constantine A. Stratakis, MD, DSc, Program in Developmental Endocrinology and Genetics, NICHD, Bethesda, MD

Contact

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

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