Alfred L. Yergey, PhD, Head, Section on Metabolic Analysis and Mass Spectrometry
Jozsef Antal, PhD, Visiting Fellow
Peter S. Backlund, PhD, Staff Scientist
Matthew Olson, BS, Predoctoral Fellow
Elizabeth Okbonna, BS, Contractor
Nancy E. Vieira, MS, Biologist

We carry out research in areas of chemistry, biochemistry, and medicine for which mass spectrometry is the primary analytical tool. Our current research focuses on the reaction energetics of gas phase ions and protein characterization.

Reaction energetics of gas phase ions

Yergey; in collaboration with Blank, Campbell, Stein, Vestal

We aim to understand the relationship between the energy applied to the formation of a gas-phase peptide ion and the nature of the ion's fragmentation. The type and extent of fragmentation are the determining factors of MS-MS spectra, and these spectra are the foundation on which mass-spectrometric characterization of proteins is based.

We are using matrix-assisted laser desorption ionization (MALDI) of peptides as a model system to study peptide ion fragmentation. The study of the ion energetics relationships between laser fluence and peptide ion fragmentation is fundamental to optimizing MALDI TOF/TOF experiments for the purpose of peptide sequencing. In our studies, we obtain peptide fragmentation spectra, typically 5,000 laser shots, in both the unimolecular decomposition and collision-induced dissociation (CID) modes. We have the ability to follow easily two time points for each peptide decomposition, i.e., in-source fragmentation consisting of ions formed within 1 µsec after laser firing and longer, mass-dependent fragmentation occurring within the instrument's collision cell. To date, we have used the fragmentation of a model peptide, leucine enkephalin (LeuEnk, YGGFL), over the full range of laser fluence. While not a peptide of the type normally encountered in protein characterizations, LeuEnk is an excellent model for studies of short-lived processes in the laser plume and has undergone extensive study with other mass-spectrometric (MS) approaches. Spectra are acquired as a function of laser fluence beginning at the onset of ionization and extending to the maximum fluence available in the instrument. We discovered earlier that the MS mode spectra show a region of extensive fragmentation occurring in what must be a very short time frame following the onset of ionization. Rapid fragmentations, leading to the observation of only immonium ions, are associated with low laser plume densities. A second set of processes takes place within the first several hundred nanoseconds following the laser pulse and occurs in the realm of much higher laser plume density. Ions formed in these processes undergo a large number of collisions with the high-temperature gases present in the laser plume and begin to fragment; fragmentations proceed in a series of consecutive reactions in which amide backbone bonds are ruptured. Finally, the MS-MS mode spectra of LeuEnk exhibit little fragmentation.

We found that our efforts to develop a kinetic model for these decompositions by using the Rice-Ramsberger-Kassel-Marcus (RRKM) formalism for gas phase kinetics do not adequately specify the conditions within the laser plume. To address these difficulties, we undertook to measure so-called "thermometer" molecules under conditions as similar as possible to those used for LeuEnk. "Thermometer" molecules are a series of substituted benzyl pyridinium salts with the important characteristic of only a single fragmentation reaction: the breaking of the N-benzyl bond. Our initial results show the expected fragmentation. We are currently attempting to rationalize our observations with the theoretical model.

Protein characterization

Antal, Backlund, Vieira, Yergey; in collaboration with Blank, Bonifacino, Caplan, Clouse-Strebel, Coorssen, Dasso, Epstein, Garland, Harnly, Harrington, Humphrey, Klein, Leppla, Love, Owens, Pacak, Porter, Robbins, Rouault, Russell, Sackett, Schuck, Zimmerberg

As a first priority, we conduct research on the MS characterization of proteins in collaboration with groups in NICHD, but we also undertake independent investigations in mass-spectrometric protein characterization. A major aspect of our work is the identification of proteins isolated in biochemical investigations of other researchers. In terms of the identification of unknown proteins, we use MS data to query genomic databases to determine whether any of the protein sequences in the databases have expected proteolytic cleavage products with theoretical masses that match the empirically determined masses of the peptides generated from the unknown. Our studies take advantage of three MS approaches: matrix-assisted laser desorption ionization (MALDI) with time-of-flight (TOF) mass analysis; liquid chromatography (LC) followed by electrospray ionization with mass analysis in an instrument capable of using fragmentation reactions to generate peptide sequences, i.e., LC-MS/MS; and MALDI followed by tandem TOF analysis for the determination of peptide sequences from fragment ion spectra. With this combination of instrumentation, we are confident that, given as little as 100 fmole for analysis, we can make a positive identification for a protein described in a database.

To improve protein characterization capabilities, we are pursuing several avenues. In particular, we developed a novel approach to providing sequence information for proteins not described in databases owing to either database error or incompleteness of splice variants, or SNPs; such incompleteness is associated most frequently with organisms with unknown or partially characterized genomes, e.g., Xenopus laevis. We recently improved the algorithms used for sequencing by adding the ability to predict internal fragment ions of the highest-scoring peptides found by de novo algorithms. With this addition, we have been able to eliminate potential ambiguities that arise from incomplete fragmentation in the observed data of a sequence.

In an area related to protein identification and sequencing, we reported major strides in characterizing the C-terminal post-translational modifications of tubulins. We recently made a substantial technical improvement in our methodology and, by using agarose instead of acrylamide gels, are now able to employ in-gel digestion methods rather than relying on solution digestions. Our analytic results have shown that bovine and rat brain tubulins appear to have indistinguishable compositions and, perhaps more interestingly, that the tubulins associated with clathrin-coated vesicles have the same tubulin composition as a homogenate of whole brain.

Another study investigated the effect of a particular post-translational protein modification. Asparagine deamidation is an important post-translational protein modification that increases as a protein ages. While deamidation can occur at all asparaginyl residues, the reaction rates in a protein can vary greatly depending on primary sequence and conformation. We used a combination of MALDI TOF and MALDI tandem TOF mass spectrometry for a quantitative determination of deamidation and the mapping of specific deamidation sites. We applied this method to the mapping of deamidation sites in the recombinant proteins Protective Antigen and Lethal Factor from Bacillus anthracis. Given that Protective Antigen is the basis for all current anthrax vaccines, a fundamental understanding of the extent of deamidation with aging of the protein is an important public health issue.

The project characterizing the protein mass fingerprints of amniotic fluid from patients who have undergone premature labor has progressed to a stage that permits a clear distinction between patients who delivered at term and those who delivered prematurely. Our work is the first successful effort to make such a differentiation and represents an important milestone. The methodology developed for the project employs comparisons of MALDI mass spectra in the range of 2-10 kDa obtained from diluted amniotic fluid samples that have been desalted and then applied directly to the MALDI sample stage. Our experimental design characterizes the variance of spectra arising from a variety of methodological factors. We have developed a mathematical/statistical approach in MatLab® to automate both ANOVA and Principal Component Analysis and to distinguish classes of samples reliably.

Collaborators

Paul Blank, PhD, Laboratory of Cellular and Molecular Biophysics, NICHD, Bethesda, MD
Juan Bonifacino, PhD, Cell Biology and Metabolism Branch, NICHD, Bethesda, MD
Jennifer Campbell, PhD, Beyond Genomics, Inc., Framingham, MA
Steven Caplan, PhD, University of Nebraska Medical Center, Lincoln, NE
Kathleen Clouse-Strebel, PhD, Center for Drug Evaluation and Research, FDA, Bethesda, MD
Jens Coorssen, PhD, University of Calgary, Calgary, Canada
Mary Dasso, PhD, Laboratory of Gene Regulation and Development, NICHD, Bethesda, MD
Jonathan Epstein, MS, Unit on Biologic Computation, NICHD, Bethesda, MD
Donita Garland, PhD, Laboratory of Mechanisms of Ocular Disease, NEI, Bethesda, MD
James Harnly, PhD, Human Nutrition Research Center, USDA, Beltsville, MD
Peter de B. Harrington, PhD, Ohio University, Athens, OH
Glen Humphrey, PhD, Laboratory of Cellular and Molecular Biophysics, NICHD, Bethesda, MD
David Klein, PhD, Section on Neuroendocrinology, NICHD, Bethesda, MD
Steven Leppla, PhD, Bacterial Toxins and Therapeutics Section, NIAID, Bethesda, MD
Paul Love, MD, PhD, Laboratory of Mammalian Genes and Development, NICHD, Bethesda, MD
Thomas Neubert, PhD, Skirball Institue of biomolecular Medicine, New York University, New York, NY
Ida Owens, PhD, Heritable Disorders Branch, NICHD, Bethesda, MD
Karel Pacak, MD, PhD, DSc, Reproductive Biology and Medicine Branch, NICHD, Bethesda, MD
Forbes Porter, MD, PhD, Heritable Disorders Branch, NICHD, Bethesda, MD
Juan Rivera, PhD, Molecular Immunology and Inflammation Branch, NIAMS, Bethesda, MD
John Robbins, MD, Laboratory of Developmental and Molecular Immunity, NICHD, Bethesda, MD
Tracey Rouault, MD, Cell Biology and Metabolism Branch, NICHD, Bethesda, MD
James Russell, DVM, Section on Cell Biology and Signal Transduction, NICHD, Bethesda, MD
Dan Sackett, PhD, Laboratory of Integrative and Medical Biophysics, NICHD, Bethesda, MD
Peter Schuck, PhD, Division of Bioengineering and Physical Science, ORS, NIH, Bethesda, MD
Douglas Sheeley, ScD, Division of Biomedical Technology, NCRR, Bethesda, MD
Stephen Stein, PhD, MS Data Center, NIST, Gaithersburg, MD
Gisela Storz, PhD, Cell Biology and Metabolism Branch, NICHD, Bethesda, MD
Akos Vertes, PhD, George Washington University, Washington, DC
Marvin Vestal, PhD, Virgin Instruments, Framingham, MA
Joshua Zimmerberg, PhD, MD, Laboratory of Cellular and Molecular Biophysics, NICHD, Bethesda, MD

For further information, contact aly@helix.nih.gov.