Researchers at the Johns Hopkins Bloomberg School of
Public Health have developed a new method for identifying
specific proteins in whole cell extracts of microorganisms
using traditional peptide mass fingerprinting, or PMF.
The key to the new method, according to the
researchers, is a "shortcut" for preparing samples that
makes PMF faster and more economical. By reducing the cost
of protein identification, they believe PMF can become an
economical tool for monitoring and evaluating the
effectiveness of microorganisms used in environmental
cleanup. The researchers used a dioxin-eating organism to
demonstrate the capabilities of their methodology, which
they described in an article published in the May edition
of Applied and Environmental Microbiology.
PMF typically involves elaborate sample preparation. A
protein mixture is spread across a gel and separated into
individual proteins, which are scooped out of the gel and
cut with protein scissors into predictable small pieces
called peptides. The samples are then analyzed using
matrix-assisted laser desorption ionization-time of flight
mass spectrometry, which identifies protein fragments based
on the time they need to travel a defined distance when
being accelerated in a vacuum.
In their study, Rolf U. Halden, assistant professor in
the Bloomberg School's
Department of Environmental Health Sciences, and his
colleagues demonstrate how PMF and mass spectrometry are
used to identify a unique dioxin-degrading enzyme in a soup
of hundreds of cell proteins. The technique avoids
elaborate conventional sample preparation steps by coaxing
the cells into mass production of the protein the
researchers wish to analyze.
"Finding a specific target in a mixture of hundreds of
proteins can be likened to finding the proverbial needle in
the haystack; this task can be performed much faster and
more economically if you have more needles--and that's
exactly what our method is based on," Halden says. "Instead
of spending a lot of time and resources on eliminating the
background noise to find the signal, our method increases
the signal up front, so that it stands out above the
background noise. By forcing an up-regulation of enzyme
expression in the bacterium of interest, our target can be
identified amidst all of the other cell components," he
says.
Halden and his colleagues tested their technique using
Sphingomonas wittichii strain RW1, the only bacterium known
to consume the backbone of toxic polychlorinated
dibenzo-p-dioxins and dibenzofurans as a food source. The
researchers already knew that feeding dioxins to RW1 would
cause an increased enzyme level as the bacterium consumed
the model pollutant. Their study shows that this increase
can be easily identified by PMF using mass spectrometry.
"Our procedure simplifies the entire identification
process," says David Colquhoun, a doctoral fellow with the
Johns Hopkins
Center for a Livable Future, "With the new tool, we can
conveniently and rapidly identify both pollutant-degrading
bacteria and their characteristic proteins that effect
pollutant transformation."
"This method represents a new investigative tool in
bioremediation, which is the science of using biological
organisms as a means of decontaminating polluted soils and
water," Halden says.
The study was written by Halden, Colquhoun and E.S.
Wisniewski. Funding was provided by grants from the Johns
Hopkins Bloomberg School of Public Health Technology
Transfer Committee, the National Institutes of Health
Training Grant and the Johns Hopkins Center for a Livable
Future.
Johns Hopkins University is seeking partners who would
like to license this patent-pending methodology. Inquiries
may be directed to Deborah Alper at the School of Public
Health at
dalper@jhsph.edu or 443-287-0402.