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applications of
mass spectrometry to biophysics,
biochemistry and
medicine
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Alfred L. Yergey, PhD, Head, Section on Metabolic Analysis and Mass
Spectrometry Jozsef Antal, PhD, Visiting Fellow Peter S. Backlund, PhD, Staff Scientist Peter deB Harrington, PhD, Contractor Matthew Olson, BS, Predoctoral Fellow Daniel Spellman, BS, Graduate Student Nancy E. Vieira, MS, Biologist |
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We carry out research in areas of chemistry,
biochemistry, and medicine in which mass spectrometry (MS) is the primary
analytic tool. Our current research focuses on the reaction energetics of gas
phase ions, protein characterization, and the quantification of endogenous
molecules. Reaction energetics of gas phase ions Yergey;
in collaboration with Blank, Campbell, Stein, Vestal The principal aim of our work is 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, which 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 and ion energetics relationships between laser fluence and
peptide ion fragmentation. Such a study is fundamental to optimizing MALDI
Time-of-Flight (TOF)/TOF experiments for the purpose of peptide sequencing.
In our studies, we obtain peptide fragmentation spectra, typically after
5,000 laser shots, in both the unimolecular decomposition and
collision-induced dissociation (CID) modes. We have the ability to follow two
time points for each peptide decomposition, i.e., the in-source fragmentation
consisting of ions formed within one microsecond after the laser firing and the
longer, mass-dependent fragmentation occurring within the instrument’s
collision cell. We have used the fragmentation of the YGGFL model peptide
(leucine enkephalin;Â LeuEnk) over the
full range of laser fluence as the basis for our initial studies. 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. We have acquired LeuEnk fragmentation spectra
in both MS and MS-MS modes of operation as a function of laser fluence
beginning at the onset of ionization and extending to the maximum fluence
available in the instrument. The spectra reveal several distinct processes in
LeuEnk fragmentation. First, the MS mode spectra show a region of extensive
fragmentation occurring in what must be a markedly short time frame following
the onset of ionization. We have been able to associate these rapid
fragmentations, leading to immonium ions, with what is widely accepted to be the
laser pulse–induced direct vaporization of molecules from the sample
surface. A second set of processes takes place within the first several
hundred nanoseconds following the laser pulse. These processes are most
likely associated with desorption of LeuEnk ions from particles ablated from
the surface. The desorbed ions undergo a large number of collisions with the
high-temperature gases present 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. Our spectra show that the initial direct
desorption processes reach a maximum and then increase no further and that
they are supplanted in intensity by the consecutive fragmentation reactions.
Finally, the MS-MS mode spectra exhibit little fragmentation, most likely
because of depletion of the high-energy portions of the energy distributions
associated with the second-stage particle desorption processes described
above. We are simultaneously developing a kinetic
model for the decompositions by using the Rice-Ramsberger-Kassel-Marcus
formalism for gas phase kinetics. In addition to modeling fragmentation, the
calculations will define a lower limit of the peptide ion temperatures. With
this information, we will be able for the first time to estimate the fraction
of laser energy delivered to gas phase ions. Furthermore, we will have a
means, other than pure empiricism, to select and optimize both the MALDI
matrix and laser frequency. Gilligan JJ, Lampe FW, Nguyen VQ, Gilligan, JJ, Protein characterization Antal,
Backlund, Harrington, Olson, Spellman, Vieira, Yergey; in collaboration with
Blank, Coorssen, Crouch, DePamphilis, Epstein, Garland, Howard, Humphrey,
Leppla, Robbins, Rouault, Sackett, Schneerson, Schuck, Sheeley, Spiegel,
Zimmerberg As our first priority, we conduct research
collaboratively with various NICHD groups on the mass spectrometric
characterization of proteins, but we also conduct independent investigations
in mass spectrometric protein characterization. A major aspect of our work is
the identification of proteins isolated in the biochemical investigations of
other investigators. To identify unknown proteins, we use the MS
data to query genomic databases to determine whether any of the protein
sequences present in those databases have the expected proteolytic cleavage
products with theoretical masses that match the empirically determined masses
of the peptides generated from the unknown. Three MS approaches are available
for this effort: MALDI with 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 sufficient material in a gel
band to allow as much as 100 fmole to be available for analysis, we can make
a positive identification for a protein described in a database. Within the past year, we started a project to
characterize the protein mass fingerprints of amniotic fluid from patients
who have undergone premature labor. We hypothesize that mass spectra can be
used to differentiate premature labor leading to pre-term delivery from such
labor that does not result in pre-term delivery. The methodology under
development employs comparisons of MALDI mass spectra in the range of 2 to
20kDa obtained from diluted amniotic fluid samples that have been desalted
and then applied directly to the MALDI sample stage. We developed an
experimental design that allows us to characterize the variance of the
spectra arising from a variety of parameters in the experiment. We have
developed a mathematical/statistical approach in MatLab to automate both
ANOVA and Principal Component Analysis and reliably differentiate between
classes of samples. Preliminary results show that we are able to
differentiate between the sources of amniotic fluid in groups of patients. Jeong HS, Backlund PS, Chen HC, Karavanov AA,
Crouch RJ. RNase H2 of Saccharomyces cerevisiae is a complex of three
proteins. Nucleic Acids Res 2004;32:407-414. Li W, Backlund PS, Boykins RA, Wang G, Chen
H-C. Susceptibility of the hydroxyl group in serine and threonine to
beta-elimination/Michael Addition under commonly used moderately high
temperature conditions. Anal Biochem 2003;323:94-102. Complete de novo sequencing of peptides
Backlund,
Yergey We are pursuing several areas of development
in order to improve protein characterization capabilities. First, we are
addressing the issue of sequence information on proteins that, as a
consequence of database error or incompleteness, are not described in
databases; the incompleteness is most frequently associated with organisms
with unknown or partially characterized genomes. We are taking an approach we
have termed Complete de Novo Sequencing of Peptides, which is novel as
compared with the other widely used methods in which the so-called “sequence
tag� for a peptide is found. The sequence tag approach consists of
determining between two and five amino acid residues from a peptide
fragmentation mass spectrum along with the parent mass, which then allows us
to search a database. Our approach requires the determination of the amino
acid sequence of the entire peptide and requires the use of a MALDI tandem
TOF. We use software we have written to analyze the MALDI tandem TOF spectra,
giving us a unique ability to sequence proteins. Our procedure relies on the
decomposition of metastable peptide ions, without subsequent collisions, in
the time frame established by the selection of candidate ions after the first
mass analyzer, but prior to the so-called “collision cell.� The method
yields highly reliable sequences for as many as seven peptides in the range
of seven to 18 residues taken from the tryptic proteolysis of proteins
analyzed as unknowns; in the case of a 30kD protein, the method would yield
an absolute determination of about 30 percent of the entire sequence of the
protein. In the past, one limitation of the approach related to near-isobaric
interferences, i.e., precursor ion masses that cannot be resolved by the mass
selection electronics, thus leading to fragmentation spectra arising from
multiple precursor ions that are unintepretable. The use of a recently
installed sample-spotting robot to enable spatial separations of LC gradients
onto a MALDI target plate should reduce the number of such interferences,
thereby allowing more extensive coverage of absolute sequences. In an area closely related to the Complete de
Novo Sequencing work, we have developed and implemented software that
increases our ability to pinpoint certain types of peptides: those containing
post-translational modifications and those arising from less commonly used
proteases, particularly pepsin. In addition, we have made substantial
progress in the detection of phosphorylation sites by using a differential
MALDI spectra approach that compares positive and negative ion spectra. The
method employs the esterification of carboxylic acid sites with methanolic
HCl; the esterified acidic residues do not ionize efficiently in negative ion
MALDI, but the phosphorylated peptides are unaffected. Publications
Related to Other Work Ben-Menachem G, Kubler-Kielb J, Coxon B,
Yergey A, Schneerson R. A newly discovered cholesteryl galactoside from Borrelia
burgdorferi. Proc Natl Acad Sci USA 2003;100:7913-7918. Buzas Z, Antal J, Gilligan JJ, Backlund PS,
Yergey AL, Chrambach A. An electroelution apparatus for sequential transfer
of dodecyl sulfate proteins into agarose and mass spectrometric
identification of Li- Na-dodecyl sulfate-proteins from solubilized agarose. Electrophoresis
2004;25:966-969. Naslavsky N, Boehm M, Backlund PS Jr, Caplan
S. Rabenosyn-5 and EHD1 interact and sequentially regulate protein recycling
to the plasma membrane. Mol Biol Cell 2004;15:2410-2422. Nelson TJ, Backlund PS, Alkon DL. Hippocampal
protein-protein interactions in spatial memory. Hippocampus
2004;14:46-57. Schneerson R, Kubler-Kielb J, Liu T-Y, Dai
A-D, Yergey A, Backlund P, Shiloach J, Leppla S, Majadly F, Robbins JB. Poly-g-D-glutamic
acid protein conjugates induce IgG antibodies in mice to the capsule of Bacillus
anthracis: a potential addition to the anthrax vaccine. Proc Natl Acad
Sci USA 2003;100:8945-8950. COLLABORATORS Paul Blank, PhD, Laboratory
of Cellular and Molecular Biophysics, NICHD, Jennifer Campbell, PhD, Applied
Biosystems, Jens Coorssen, PhD, Robert Crouch, PhD, Laboratory
of Molecular Genetics, NICHD, Melvin DePamphilis, PhD, Laboratory
of Molecular Growth Regulation, NICHD, Jonathan Epstein, MS, Office
of the Scientific Director, NICHD, Donita Garland, PhD, Laboratory
of Mechanisms of Ocular Diseases, NEI, Bruce Howard, MD, Laboratory
of Molecular Growth Regulation, NICHD, Glen Humphrey, PhD, Laboratory
of Cellular and Molecular Biophysics, NICHD, Steven Leppla, PhD, Bacterial
Toxins and Therapeutics Section, NIAID, Sergey Radko, PhD, Laboratory
of Cellular and Molecular Biophysics, NICHD, John Robbins, MD, Laboratory
of Developmental and Molecular Immunity, NICHD, Tracey Rouault, MD, Cell
Biology and Metabolism Branch, NICHD, Dan Sackett, PhD, Laboratory
of Integrative and Medical Biophysics, NICHD, Rachel Schneerson, MD, Laboratory
of Developmental and Molecular Immunity, NICHD, Peter Schuck, PhD, Division
of Bioengineering and Physical Science, Office of Research Services, NIH, Douglas Sheeley, ScD, Division
of Biomedical Technology, NCRR, Alan Spiegel, MD, Director,
NIDDK, Stephen Stein, PhD, NIST
MS Data Center, Akos Vertes, PhD, Marvin Vestal, PhD, Applied
Biosystems, Joshua Zimmerberg, PhD, MD, Laboratory
of Cellular and Molecular Biophysics, NICHD, For
further information, contact aly@helix.nih.gov |
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