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Mass Spectrometry Core Facility

Alfred L. Yergey, PhD
  • Alfred L. Yergey, PhD, Principal Investigator, Co-Director
  • Peter S. Backlund, PhD, Staff Scientist, Co-Director
  • Elizabeth Ogbonna, BS, Contractor
  • Nancy E. Vieira, MS, Senior Research Assistant

The NICHD Mass Spectrometry Core Facility was created under the auspices of the Office of the Scientific Director to provide high-end mass-spectrometric services to all NICHD scientists, particularly in the area of protein characterization. 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 to everyone within the institute free of charge, provided that existing resources are distributed equally among investigators requesting our services. Except for the ABI Voyager MALDI TOF (MALDI: matrix-assisted laser desorption/ionization; TOF: time-of-flight) instrument, none of the equipment is available for general use. The philosophy of the Mass Spectrometry Facility is to ensure that its instruments record only reliable, high-quality data and that its clients receive only statistically meaningful analyses. For every new project, staff members meet with the Principal Investigator (PI) and the postdoctoral scientists involved in the study to discuss details of the experimental design. The discussion focuses on the project’s background and analytical goals in order to determine the most appropriate techniques for sample preparation before delivery of samples to the laboratory. We digest the proteins, typically gel-separated by the investigator, in situ and isolate peptides for mass-spectrometric analysis. We subject separated peptides to analysis by fragmenting them in a second stage of mass spectrometer operation and then use the fragmentation spectra as input for database search engines to determine the protein(s) present in the original sample. Upon completion of an analysis, we provide the investigators with the results and review the findings. Long-term user feedback indicates that our high level of interaction with researchers has greatly improved the quality and utility of results of each project undertaken.

Instrumentation

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

  1. ABI 4800 MALDI TOF/TOF. This state-of-the-art high-performance instrument can be operated in both positive- and negative-ion modes. We typically use it for peptide mixtures without chromatographic separation. Additional uses include accurate determination of peptide masses in complex mixtures and the determination of sequences of unusual peptides through de novo sequencing techniques.
  2. Thermo Finnigan LCQ Deca
  3. LCQ DecaXP. Both the thermo Finnigan lcQ deca and the lcQ decaXp employ liquid chromatography and electrospray ionization to separate and ionize peptides that are then fragmented. At present the DecaXP instrument is dedicated to use in a project of cardiolipid quantification in serum.
  4. ABI Voyager MALDI TOF. This instrument is used for analysis of higher–molecular-weight species—equal to or greater than 125 kDa—and to verify the molecular weight of intact proteins. The instrument is also available for general use after a potential user has undergone the appropriate training.

Facility usage

The Mass Spectrometry Facility currently serves 15 sections within the institute, representing about 40 projects, as well as four PIs of sister institutes on the NIH campus.

Major projects during Fiscal Year 2008

Brain proteomics in Smith-Lemli-Opitz syndrome (SLOS). For this investigation, investigators use mouse models of human genetic diseases involving defects in cholesterol biosynthesis. Proteomic studies of wild-type and knockout mouse (Dhcr7 and Sc5d) brain tissue, using image analysis of two-dimensional gels, have led to the determination of 64 proteins with expression differences exceeding 1.5-fold. For protein identification, at least three peptides were matched with greater than 95% confidence, and total amino acid sequence coverage ranged from 14% to 74% of the total protein. HMG CoA synthase is an example of a differentially expressed protein observed in brain extracts. Levels of HMG CoA synthase in brains of knockout animals were significantly higher than in wild type, with ratios of control to knockout of 1:1.55 for Dhcr7, and 1:1.75 for Sc5d. Given that HMG CoA synthase is the first step in the cholesterol biosynthetic pathway, a reduction in brain cholesterol levels would be expected to increase the activity/expression of this enzyme, which was observed for both knockouts. In addition to changes in the level of protein expression, this approach also detected changes in protein post-translational modifications in brain proteins from knockout mice. A shift in protein mobility on the gel permitted detection of these modifications. An example of this change was observed for cofilin 1. Two gel spots were observed to contain cofilin 1, and the spot migrating with a more acidic isoelectric point was more intensely stained in knockout mice than in wild type controls. Western Blots, using an anti-phospho cofilin 1 antibody, confirmed the increase in cofilin 1 phosphorylation. Another example of a shift in protein migration was observed for the Rab7 protein. Rab 7 is a member of a large family of GTP-binding proteins. Many of the proteins in this family are modified by prenylation at C-terminal cysteine residues. Protein prenylation is a side branch in the biochemical pathway for cholesterol biosynthesis, and alterations in cholesterol biosynthesis could also result in changing the levels of protein prenylation. Prenylation is known to shift the mobility of at least some GTP-binding proteins, and the data suggest that a change in Rab7 prenylation may result in the observed mobility shift. However, a direct determination of Rab7 prenylation was not performed here, and other post-translational modifications may also be involved.

Lipid quantification in serum. We continue to develop methodology to quantify cardiolipins in human serum by mass spectrometry. This effort is associated with a clinical study under way in the Institute to evaluate the effects of antibiotic treatment of pregnant women colonized with the Group B streptococcal (GBS) organism. The hypothesis of the study is that the typical peri-natal penicillin treatment gives rise to a large increase of circulating cardiolipins in the infant, which leads to respiratory distress. It has been demonstrated in newborn sheep that, upon treatment with penicillin, the GBS organisms secrete a specific cell wall membrane cardiolipin, which causes respiratory distress at levels corresponding to the injection of about 100 pmole/mL in serum; this concentration falls rapidly with a half life of a few minutes. It is not known whether the respiratory distress observed in a fraction of infants born to GBS colonized mothers is a result of a similar effect, or caused by the release of endogenous cardiolipins stimulated by bacterial death. The analytical approach involves the addition of an internal standard to a serum sample, extractions by a combination of liquid-liquid and solid phase, followed by an LC-MS (LC liquid chromatography; MS: mass spectrometry) analysis that incorporates an extraction/recovery standard to provide system quality control. We have shown that cardiolipin can be extracted from serum with approximately 90% efficiency. We have further shown that normal adult levels of (18:2)4 cardiolipins are present in serum at levels of less than 10 fmole/µL, which is approximately 1000-fold lower than found by earlier, less accurate measurements. With this base-line information, we are seeking to establish the range of normal serum levels in cord blood of mothers not colonized by GBS and will then determine levels in infected individuals.

Quantification of histone demethylation. Histone methylation regulates chromatin structure, transcription, and the epigenetic state of the cell. Histone methylation is dynamically regulated by histone methylases and demethylases, which mediate demethylation of di- and monomethylated histones. It has been unclear whether demethylases exist that can reverse lysine trimethylation. A putative histone demethylase, KDM3B, that was identified in earlier investigations, is being investigated to probe the specificity and extent of its demethylation capability, using a model peptide synthesized with a lysine residue containing 1, 2, or 3 methylations. The methylated peptide ARTKQTAR[K]STGGKAPRKQLAGGK-biotin is incubated with either immuno-precipitated (IP) or recombinant KDM3B; the ratio of de-methylated product to substrate is determined by MALDI/TOF. Replicate mass-spectral measurements of the peptide substrate from control and enzyme experiments, both with and without the putative co-factor RanQ69L-GTP, are corrected for background levels, and the fractional de-methylation calculated. Initial studies with recombinant KDM3B showed no activity, while the IP enzyme showed activity for de-methylation of 1-Me, removal of both methyl groups from 2-Me and no activity with the 3-Me substrate. A Perl script has been written to permit processing of multiple replicate MALDI-TOF mass spectral peak lists, extract peak intensities, and calculate fractional demethylation.

Improvement in spectral reliability. We have continued to make advances in our efforts to develop methods for obtaining more robust mass spectra of samples by generating consensus spectra from several replicates. When applied to peptide fragmentation spectra as part of an effort to infer the presence of proteins in gel samples, our approach—particularly when applied to peptide sequencing de novo—shows substantial improvement, as reflected in our preliminary results. Our novel algorithm for deducing peptide sequences de novo often yields useful results for a particular peptide precursor undergoing fragmentation and appears to yield meaningful results with each application, generating a consensus spectrum from several replicate spectra of the same peptide precursor. Accordingly, we plan to extend the approach to routine protein inference from unknown samples in both MALDI TOF-TOF and LC/MS-MS methodologies.

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 the isolation and staining of gels and 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.

Publications

  • Yun C, Wang Y, Mukhopadhyay D, Backlund P, Kolli N, Yergey A, Wilkinson K, Dasso M. Nucleolar protein B23/nucleophosmin regulates the proteases. J Cell Biol 2008 183:589-595.
  • Olson MT, Blank PS, Sackett DL, Yergey AL. Evaluating reproducibility and similarity of mass and intensity data in complex spectra—applications to tubulin. J Am Soc Mass Spect 2008 19:367-374.
  • Olson Matthew T, Yergey Alfred L. Calculation of the isotope cluster for polypeptides by probability grouping. J Am Soc Mass Spect 2009 20:295-302.
  • Duncan MW, Yergey AL, Patterson SD. Quantifying proteins by mass spectrometry: the selectivity of SRM is only part of the problem. Proteomics 2009 9:1124-1127.

Contact

For more information, email aly@helix.nih.gov.

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