Scientific Presentations Summer 2009 - Dana-Farber/Harvard ...

High-throughput Methods for Human Interactome Network Mapping
Nancy Rivera
Mentors: David E. Hill, PhD and Marc Vidal, PhD
Scientific Advisors: Matija Dreze and Pascal Braun, PhD
Dana-Farber Cancer Institute
Protein-protein interactions (PPI) are of central importance to the functioning of
cells. Thus, studying PPI is essential to improve our understanding of basic biology,
organism development and diseases. Advancements in technology have allowed researchers
to progress from studying one unique PPI at a time to global investigations
of hundreds to thousands of PPI at a time. These advancements will eventually lead
to a better understanding of disease processes and more rapid development of therapeutics,
particularly for cancer. In order to evaluate the quality of PPI datasets, we
are developing a high throughput validation system based on testing each interaction
pair using multiple, distinct assays having empirically determined assay sensitivities
and specifities. To calibrate each of the assays with respect to their inherent false
negative and false positive rates, two reference sets of interaction pairs have been
assembled. The positive reference set (PRS) is comprised of 100 well-documented
pairs of interacting human proteins while a random reference set (RRS) contains 100
pairs in which each partner is randomly chosen from a collection of ~15,000 individual
proteins. The two reference sets are then tested in all assays to establish assay
parameters. Any given interaction pair from a PPI dataset is then tested in each of
the distinct assays and the results are combined to derive a “confidence score” for each
interaction pair. Affinity purification assay using Glutathione S-transferase (GST)
pulldowns followed by western blotting has been traditionally used for confirming
PPI but overall assay sensitivity and specificity has not been established. We now are
defining the assay parameters for GST pull-downs by first applying it to the PRS/
RRS sets. GST pull-down results for the PRS/RRS compare favorably to those seen
for other assays, albeit with some interesting differences that will be discussed.