The Section on Metabolic Analysis and Mass Spectrometry carries out research
in areas of chemistry and biochemistry in which mass spectrometry is the
primary analytical tool. Current research focuses on two separate areas:
the energetics of hydration and protein characterization. The goal of
this fundamental physicochemical research is to provide missing, critical
information needed for the biophysical characterization of macromolecular
and membrane interactions and a characterization of the energy required
for noncovalent bonds between molecules or ions in solution. Much of the
current activity in theoretical studies of molecular configuration is
utterly dependent on the values assigned to solute/solvent or peptide/metal
bonds, yet reliable quantities often do not exist. By adapting mass spectrometric
concepts on cluster formation in systems of small ions and molecules,
we are investigating the energetics of the interactions of some model
solutes, as well as of amino acids and small model peptides, with water.
Hydration Thermodynamics
Vieira, Gilligan, Yergey
In the first area, we are determining the energy required to form and
break noncovalent bonds between molecules and ions in solution. The particular
bonds of interest are those involved in the interplay with water of solutes,
including amino acids, small peptides, and lipids. The objective of our
thermodynamic investigations is to determine solvation enthalpies and
free energies of selected biologically interesting ions. Knowledge of
the energetic requirements of these noncovalent bonds, particularly those
involving water and biologically significant molecules, is fundamental
to understanding molecular interactions and the changes in conformations
that are integral to them.
Experimentally, we use the tools of equilibrium gas phase ion-molecule
chemistry applied in a modifed electrospray ionization (ESI) source of
a single quadrupole mass filter. In this work, we determine the thermodynamic
quantities deltaH(std)298, deltaS(std)298, and deltaG(std)298 by using
the approach of equilibrium ion-molecule reaction chemistry. We calculated
hydration thermodynamics values from equilibrium constants measured over
a temperature range of 0� to136�C
at ion source and water partial pressures ranging between zero and 100
mtorr. We made equilibrium ion intensity measurements for at least four
hydration states, i.e., zero through three water molecules associated
with a core ion, at each of at least 60 combinations of water partial
pressures and temperatures covering the ranges of experimental variables.
The initial goal of our work is to determine the enthalpy of solvation
of a series of alkylammonium ions, CnH(2n+1)NH3+. While the ions are clearly
a model system, they offer the possibility of providing insights into
the understanding of the relationship between hydrocarbon chain length
and solvation as well as the hydration of lipids and membranes. Previous
researchers determined thermodynamic parameters for about 25 percent of
the hydrations we have studied, and results from our measurements are
in close agreement with the published data. In the past year, we completed
our measurements in the model system.
We have begun extending our work to biologically more significant systems.
Observations by several investigators have shown different effects of
1,2-(OH)2-propane and 1,3-(OH)2-propane on membrane fusion and collagen
self-assembly. It has been hypothesized that the differential effects
might be attributed to differences in the organization of water around
the two molecules. Our equilibrium ion molecule studies of these two simple
diols points to substantial differences in their hydration thermodynamics.
The step-wise addition of water to protonated 1,2-(OH)2-propane shows
a trend of diminishing exothermicity for each addition. While we were
unable to obtain a direct measurement for the addition of the first water,
we were able to estimate an upper limit on the exothermicity for this
process based on the water partial pressure and temperature. We conclude
that the first hydration step is energetically very favorable. The decreasing
trend in energetics seen for 1,2-(OH)2-propane was observed for the addition
of the first two water molecules to 1,3-(OH)2-propane but was not maintained
for the addition of the third water molecule. The addition of the third
water to the complex was determined to be energetically more favorable
than the addition of the second and furthermore was found to have a substantial
decrease in the entropy for the process. These two observations led to
our conclusion that the 1,3-(OH)2-propane trihydrate is an energetic and
entropic favorable state. The existence of such a favorable state of hydration
is consistent with the 1,3-diol incorporating into and disrupting otherwise
stable biomolecular structures.
Protein Characterization
Backlund, Li, Gilligan, Vieira, Yergey
In the second area of interest, we conduct research on the mass spectrometric
characterization of proteins. We carry out this work collaboratively with
groups in NICHD as our first priority but also conduct independent investigations
of mass spectrometric protein characterization. A major aspect of our
work is the identification of proteins isolated in biochemical investigations.
In terms of the identification of unknown proteins, we use mass spectrometric
data to query genomic databases to ask whether any of the protein sequences
present in the database have expected proteolytic cleavage products with
theoretical masses that match the empirically determined masses of the
peptides generated from an unknown sample. We use both Matrix Assisted
Laser Desorption Ionization (MALDI) with Time-of-Flight (TOF) mass analysis,
and liquid chromatography followed by electrospray ionization with mass
analysis in an instrument capable of using fragmentation reactions to
generate peptide sequences, i.e., MS/MS. With the latter method, we can
identify proteins in mixtures at levels of about 100 fmole when they are
injected into packed capillary reverse phase columns operated at 400 nL/min
flow rates. With these approaches, we are confident that, given a minimum
of 100 fmole to be applied to the LC column from a gel band, we can make
a positive identification for a protein that is described in a database.
Two principal areas of development will improve our protein characterization
capabilities. First, we have begun addressing the question of providing
sequence information on proteins that are not described in databases because
of either gaps or errors in the database. We are taking the approach termed
de novo sequencing of peptides; it requires detailed interpretation of
individual mass spectra of peptides that have been subjected to fragmentation
and mass analysis of the fragments. We are using a recently developed
tandem TOF mass spectrometer in conjunction with interpretation software
written by this section. To date, we have been reasonably successful in
applying the approach to the sequencing of proteins isolated from sea
urchin cortical vesicles (CV). Specifically, we have shown that components
of several bands isolated from the CV of sea urchins have sequences corresponding
to no known proteins yet demonstrate homologies to other proteins that
may provide insights into fusion processes.
The second area of development involves the use of surface plasmon resonance
detectors, as implemented by Biacore instruments, to isolate specific
proteins. We have developed a model system in which we have demonstrated
the isolation of 30 fmole of hen egg lysozyme in the presence of 30 pmoles
of myoglobin. The isolated proteins are then recovered in small volumes
so that concentrations are appropriate for mass spectrometric analysis.
MALDI analysis of the recovered proteins shows strong signals from the
isolated material and zero response from other proteins present.
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PUBLICATIONS
- Blank
PS, Sjomeling CM, Backlund PS, Yergey AL. Use of cumulative distribution
functions to characterize mass spectra of intact proteins. J Am Soc
Mass Spectrom 2001, in press.
- Colvis
CM, Duglas-Tabor Y, Werth KB, Vieira NE, Kowalak JA, Janjani A, Yergey
AL, Garland DL. Tracking pathology with proteomics: identification
of in vivo degradation products of aB-crystallin. Electrophoresis 2000;21:2219-2227.
- Mauras
N, O Brien KO, Welch S, Rini A, Helgeson K, Vieira NE, Yergey AL.
Insulin-like growth factor I and growth hormone (GH) treatment in GH-deficient
humans: differential effects on protein, glucose, lipid, and calcium
metabolism. J Clin Endocrinol Metab 2000;85:1686-1694.
- Praetorius
J, Backlund P, Yergey AL, Spring KR. Specific lectin binding to
beta-1 integrin and fibronectin on the apical membrane of Madin-Darby
canine kidney cells. J Membrane Biol 2001;184:273-281.
- Vieira
NE, Goans RE, Weiss GH, Marini JC, Hopkins E, Yergey AL. Calcium
kinetics in children with osteogenesis imperfecta type III and IV: pre
and postgrowth hormone therapy. Calcified Tissue Int 2000;67:97-100.
- Yefimov,
S, Sjomeling C, Yergey AL, Chrambach A. Stacking of unlabeled sodium
dodecyl sulfate-proteins with fluorimetrically detected moving boundary,
electroelution and mass spectrometric identification. Electrophoresis
2001;22:999-1003.
- Yefimov
S, Sjomeling C, Yergey AL, Li T, Chrambach A. Recovery of sodium
dodecyl sulfate-proteins from gel electrophoretic bands in a single
electroelution step for mass spectrometric analysis. Anal Biochem 2000;284:288-295.
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