Mass spectrometers, which separate molecules based
on their mass-to-charge ratio, can help researchers identify compounds
based on their unique mass and are routinely used to determine the
weight, structure and amount of small molecules or fragments of
molecules. Conventional instruments, however, are not equipped to
sensitively characterize large molecules over 150 kiloDaltons (a
measure of mass) at a low-charge state.
Using a Macromizer� mass spectrometer, Bier�s group
successfully analyzed the outer shell of the HK97 virus. They
collected a mass spectrum of the mature protein shell, which weighs
12.9 megaDaltons (12,900 kiloDaltons) and the uncleaved protein shell
(17.7 megaDaltons), which revealed an unprecedented 30+ positive
charges. They also collected an improved mass spectrum of a von
Willebrand factor (0.2 to 1.1 megaDaltons), a protein complex in blood
necessary for proper coagulation. The ability to directly mass-analyze
these heavy biological molecules intact and at a low-charge state
represents a new level of analysis previously unattainable using
conventional detector technology, according to Bier.
Many biological molecules are too big to be
analyzed efficiently at low-charge states using current mass
spectrometers, so most scientists break proteins down into smaller
fragments before analyzing them in the mass spectrometer. Although an
effective and powerful technique, this bottom-up approach typically
takes days to complete and does not allow scientists to use mass
spectrometers to directly study many large, intact proteins and other
macromolecular complexes.
Bier conducted his studies using a top-down
approach of the intact complex using a cryodetector-based MALDI TOF
mass spectrometer (Macromizer) equipped with 16 superconducting tunnel
junctions. Carnegie Mellon houses the only two of these instruments in
the U.S. Bier�s group can use the Macromizer to measure the molecular
weight of a large, intact protein or a protein complex in a matter of
seconds. Because it can measure intact protein complexes, this
approach also avoids the sample loss that typically occurs during the
bottom-up approach.
�Our results are a first step toward our ultimate
goal - to identify a virus, clotting factor or
any type of large biological molecule by just weighing it or its
gas-phase-generated fragments,� said Bier. �This would provide a rapid
clinical tool to diagnose a viral infection or a blood disease, for
example.�
Bier is collaborating with Roger Hendrix, a
professor of biological sciences at the University of Pittsburgh, who
studies how the outer shell of the HK97 virus assembles. Because
Hendrix characterizes viral proteins, particles and subunits that are
too heavy to study using currently available mass spectrometers, Bier
hopes that his data will help them discover new biology. Bier is also
collaborating with Dominic Chung, a research professor in the
Department of Biochemistry at the University of Washington in Seattle,
and Tom Howard, a medical doctor at the VA Greater Los Angeles
Healthcare System, who together study von Willebrand factors. �Mark�s
analysis by mass spectrometry displays a lot of the details about the
composition of normal human plasma von Willebrand factor. This
spectrum is truly amazing and very revealing,� Chung said.
Bier�s current work is part of a grant from the
National Science Foundation�s (NSF) Biological Infrastructure program,
which supports varied activities that provide the infrastructure for
contemporary research in biology. With this NSF support, Bier is also
building a next-generation heavy ion mass spectrometer. |