APPLICATIONS OF MASS SPECTROMETRY TO BIOPHYSICS, BIOCHEMISTRY, AND MEDICINE
, 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 (MS) 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
We aim to understand the relationship between the energy applied to the formation of a gas phase peptide ion and the nature of its fragmentation. The type and extent of the ion’s fragmentation determine the MS-MS spectra, which provide the foundation for the mass-spectrometric characterization of proteins.
We are using matrix-assisted laser desorption ionization (MALDI) of peptides as a model system to study peptide ion fragmentation and ion energetics relationships between laser fluence and peptide ion fragmentation. Such a study is fundamental to optimizing MALDI TOF/TOF (time-of-flight) experiments for the purpose of peptide sequencing. Through our studies, we obtain peptide fragmentation spectra, resulting typically from 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: (1) the in-source fragmentation consisting of ions formed within 1 µsec after laser firing and (2) the longer, mass-dependent fragmentation occurring within the instrument’s collision cell. To date, we have used the fragmentation of a model peptide, leucine enkephalin (YGGFL, LeuEnk) over the full range of laser fluence. While not a peptide of the type normally encountered in protein characterizations, LeuEnk is an excellent model that has undergone extensive study by other mass-spectrometric approaches and is appropriate for the study of short-lived processes in the laser plume. We acquire spectra as a function of laser fluence beginning at the onset of ionization and extending to the maximum fluence available in the instrument. We previously demonstrated 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. The rapid fragmentations, leading to the observation of immonium ions only, are associated with low laser plume densities. A second set of process takes place within the first several hundred nanoseconds following the laser pulse and occurs in the realm of much higher laser plume density. The ions formed in these processes undergo several collisions in the presence of the high-temperature gases in the laser plume and begin to fragment; the fragmentations proceed in a series of consecutive reactions in which the amide backbone bonds are ruptured. Finally, the MS-MS mode spectra of LeuEnk exhibit little fragmentation.
We conducted our initial studies by using a laser-pulse of 5 nsec duration. As summarized above, the studies showed that the extent of fragmentation varied with laser fluence; that is, the total extent of fragmentation increased as fluence increased, and the fragments appeared in an order consistent with the sequence of amino acids in the peptide. The sequential fragmentation occurred from both the amino and carboxy terminals of the peptide. In more recent studies, we investigated the same phenomena by using a laser-pulse length of 0.6 nsec duration and determined the fragmentation in each of three common MALDI matrices: alpha cyano-4-hydroxy cinamic acid (ACHA); 3,5-dimethoxy-4-hydroxy cinamic acid (Sinapinic acid, SA); and 2,5-dihydroxy benzoic acid (DHB). In the latter two matrix materials, we observed very little fragmentation, as might be expected from matrices widely considered to be “cooler” than ACHA, but DHB consistently showed more extensive fragments than SA. What was more surprising, however, was that the onset of extensive fragmentation in ACHA occurred at laser fluences about 50 percent lower than that observed using the 5 nsec pulse-length laser. These observations imply that, in terms of photons/unit area/unit time, the concentration of energy on the solid surface undergoing MALDI has a dominant influence on the extent to which fragmentation is observed in spectra.
Campbell JM, Stein SE, Blank PS, Epstein JA, Vestal ML, Yergey AL. Fragmentation of leucine enkephalin as a function of laser fluence in a MALDI TOF-TOF. J Am Soc Mass Spectrom 2007;18:607-16.
Protein characterization
We developed a novel approach to providing sequence information for proteins that are not described in databases because of database error, incompleteness, splice variants, or single-nucleotide polymorphisms; incompleteness is most frequently associated with organisms whose genomes are either unknown or partially characterized, e.g., Xenopus laevis. We recently modified our published approach by using an improved algorithm based on a graph theory methodology; the modification enables us to limit to a 500 Da range the size of the LIPCUT (length incremented peptide composition lookup table) database, which is the exhaustive listing of all peptide mass combinations, thereby increasing computational speed by a factor of 10. We have also extended the capability of the program to account not only for internal fragments in the observed spectra but also to include many post-translational modifications of the peptides being sequenced.
In an area related to protein identification and sequencing, we are characterizing the C-terminal post-translational modifications of tubulins. In addition to the advances in sample preparation previously reported, we recently formulated a means of developing reliable spectra from several replicate measurements. Our approach is based on the formation of a consensus spectrum and the calculation of a mean-centered spectrum from several replicates. We have improved on existing library formation methods by recognizing that a vector dot product, when used to assess similarity, can be assigned a statistical confidence level of 95 percent with the use of the Fisher Z-transform. This approach has enabled us to make a robust comparison of tubulin isotype expression in post-translational modification patterns in rat and cow brain tubulin. In addition, the approach is generally applicable to any mass-spectral data and should result in the generation of more reliable spectra for a number of purposes. Thus, we have applied the approach to replicate peptide fragmentation spectra and demonstrated that it leads to more reliable determination of de novo sequences from the resultant consensus spectrum.
In an area related to characterization of proteins’ post-translational modifications, we developed a methodology for quantifying site-specific tyrosine phosphorylation. Using the model system of activated high-affinity IgE receptors in an electrospray ionization mass spectrometer, we carried out the quantification by using a set of external peptide standards. The binding of IgE to high-affinity IgE receptors on mast cells is the initial step in the activation of signaling pathways that initiate an inflammatory response. It has been demonstrated that the Src family of protein tyrosine kinases is required for such activation. However, the detailed molecular mechanism has not been elucidated. The gamma subunit of the IgE receptor contains two tyrosine residues near the carboxyl terminus that are potential sites for phosphorylation. Site-directed mutation of each tyrosine demonstrated that both tyrosines can be substrates for phosphorylation. Based on the phosphopeptides observed in tryptic digests, we synthesized peptides with either phospho-Tyr or Tyr at the appropriate sites. We used synthetic peptides corresponding to both candidate regions to generate standard curves in order to calculate (1) relative ionization responses for phospho-Tyr and Tyr and (2) the stoichiometry for phospho-Tyr at each site. Using standard curves for each pair of standards and measuring the ratio of phosphorylated to unmodified peptide for each of the candidate tyrosine residues, we demonstrated that Tyr65 was phosphorylated at a significantly higher level than Tyr76 in the activated IgE receptor gamma subunit. While we used the method for this specific system, we recognize that it has general applicability for characterizing phosphorylation stoichiometry.
In an area unrelated to proteins, we have been developing a methodology for using MS to quantify cardiolipins in human serum. The effort is associated with a clinical study under development by the Institute to evaluate the effects of antibiotic treatment of pregnant women with group B streptococcal (GBS) infections. Our hypothesis posits that typical perinatal penicillin treatment gives rise to a large increase of circulating cardiolipins in the infant, which then leads to respiratory distress. Studies in fetal sheep have demonstrated that the GBS organisms secrete a specific cell-wall membrane cardiolipin upon penicillin treatment and that the substance causes respiratory distress. It is not known whether the respiratory distress observed in a fraction of human infants born to GBS-colonized mothers is a result of a similar effect or is perhaps caused by a release of endogenous cardiolipins stimulated by the bacterial death. We have developed cardiolipin extraction procedures for small quantities—about 20 µL—of serum in recognition of the limited sample sizes that will be available from infant cord blood at delivery. We have developed appropriate quantification standards that, in conjunction with a “standard additions” approach, will permit quantification. In addition, we have developed a reliable method for introducing the standard into a serum sample and have shown that cardiolipin may be extracted from serum with about 75 percent efficiency. At present, we are refining the mass-spectrometric methods for determination of these materials.
Backlund PS, Yergey AL. Mass spectrometry for studying protein modifications and for discovery of protein interactions. In: Schuck P, ed. Protein Interactions: Biophysical Methods for Complex Reversible Systems. Protein Reviews (Atassi Z, series ed). Springer, 2007;143-68.
Butt RH, Lee MWY, Pirshahid SA, Backlund PS, Wood S, Coorssen JR. An initial proteomic analysis of human preterm labour: placental membranes. J Prot Res 2006;5:3161-72.
Huang KP, Huang FL, Shetty PK, Yergey AL. Modification of protein by disulfide S-monoxide and disulfide S-dioxide: distinctive effects on PKC. Biochemistry 2007;46:1961-71.
Olson MT, Epstein JA, Yergey AL. De novo sequencing of peptides. In: Vekey K, Telekes A, Vertes A, eds. Medical Applications of Mass Spectrometry. Elsevier, 2008;195-202.
Reddy GS, Omdahl JL, Robinson M, Wang G, Palmore GTR, Vicchio D, Yergey AL, Tserng K-Y, Uskokovic MR. 23-carboxy-24,25,26,27-tetranorvitamin D3 (calcioic acid) and 24-carboxy-25,26,27-trinorvitamin D3 (cholacalcioic acid): end products of 25-hydroxyvitamin D3 metabolism in rat kidney through C-24 oxidation pathway. Arch Biochem Biophys 2006;455:18-30.
COLLABORATORS
Paul Blank, PhD, Program in Physical Biology, NICHD, Bethesda, MD
Juan Bonifacino, PhD, Cell Biology and Metabolism Program, 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, Program in Cellular Regulation and Metabolism, NICHD, Bethesda, MD
Jonathan Epstein, MS, Scientific Software and Bioinformatics Core Facility, 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
David Klein, PhD, Program in Developmental Endocrinology and Genetics, NICHD, Bethesda, MD
Steven Leppla, PhD, Bacterial Toxins and Therapeutics Section, NIAID, Bethesda, MD
Paul Love, MD, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
Thomas Neubert, PhD, Skirball Institute of Biomolecular Medicine, New York University, New York, NY
Ida Owens, PhD, Program in Developmental Endocrinology and Genetics, Bethesda, MD
Karel Pacak, MD, PhD, DSc, Program in Reproductive and Adult Endocrinology, NICHD, Bethesda, MD
Forbes Porter, MD, PhD, Program in Developmental Endocrinology and Genetics, NICHD, Bethesda, MD
David Proud, PhD, University of Calgary, Calgary, Canada
Juan Rivera, PhD, Molecular Immunology and Inflammation Branch, NIAMS, Bethesda, MD
John Robbins, MD, Program in Developmental and Molecular Immunity, NICHD, Bethesda, MD
Tracey Rouault, MD, Program in Molecular Medicine, NICHD, Bethesda, MD
James Russell, DVM, Program in Developmental Neuroscience, NICHD, Bethesda, MD
Dan Sackett, PhD, Program in Physical Biology, 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 Program, NICHD, Bethesda, MD
Joshua Zimmerberg, PhD, MD, Program in Physical Biology, NICHD, Bethesda, MD
For further information, contact aly@helix.nih.gov.
