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“Finding so many at one time is a huge advance,”
says Akhilesh Pandey, M.D., Ph.D., an associate professor at the
McKusick-Nathans Institute of Genetic Medicine at Hopkins.
“Phosphorylation is essential for controlling chemical reactions in
our cells’ protein factories, and phosphorylation gone awry has been
implicated in several diseases. The ability to study more than one
phosphorlyation at a time will help us understand some of these
diseases - including cancers - sooner.
“What we have here is about 20 years’ worth of lots
of work in one searchable list,” says Pandey. A report on all of the
newly identified protein alterations is published in the Feb. 13 issue
of the Proceedings of the National Academy of Sciences, while a report
on the database appears in the March issue of Nature Biotechnology.
Pandey’s team used electron transfer dissociation (ETD)
tandem mass spectrometry, a technology that breaks apart proteins into
small fragments, separates them by size and identifies the fragments
based on their mass - their size and weight. The process improves on
previous techniques by breaking up proteins more gently and keeping
chemical modifications like phosphorylation intact. Previous
spectrometry methods were “just too rough” on the delicate protein
alterations and sheared them right off, he says. “We had to guess
where they might be and nobody wants to chase false leads based on
wrong guesses.”
Pandey says the original goal of the research was
to identify accurately as many protein changes as possible using the
new technology. “But to see how well we measured up, we had to compare
our findings to what already was published, and there was just no
clean, easy way of doing that because there were reports all over the
place.
“That’s when we decided to go through and
consolidate just about everything on phosphorylation that was out
there.”
Working with human kidney cells, the researchers
fished out the thousands of different proteins and analyzed them by
ETD, resulting in a net total of 1,435 phosphorylations. Comparing
these 1,435 to the 20 years’ of published data, they discovered that
about 80 percent of what they found never had been reported.
The team then constructed an online search tool,
PhosphoMotif Finder, which was incorporated into their previously
established Human Protein Reference Database. Human Protein Reference
Database now contains about 16,000 phosphorylation sites described in
the literature and the PhosphoMotif Finder tool allows any researcher
to find potential phosphorylation sites in any protein of interest.
“The power of this technique is not just in the
numbers,” says Pandey. “Rather, we’ve found what you might call new
information about old proteins, and we hope the new data will help
researchers study their favorite proteins in greater depth. After all,
there’s no sense in reinventing the wheel.”
Pandey and his team now are curious about other
chemical modifications of proteins, which are the “business-end”
products of our genes. “There is evidence of other, more fragile
modifications that until now no one has been able to get a handle on
because they’re way too hard to work with. Now we have the tools to
probe further,” he says. |
