A complex molecule and snake venom may provide researchers with a more reliable method of diagnosing human diseases and developing new drugs.
Purdue University researchers bound a complex nanomolecule, called a dendrimer, with a glowing identification tag that was delivered to specific proteins in living venom cells from a rattlesnake. The scientists want to find a better way to ascertain the presence, concentration and function of proteins involved in disease processes. They also hope the new method will facilitate better, more efficient diagnosis in living cells and patients.
Most diagnostic methods must be done on minute dead blood or tissue cell samples in a laboratory dish, said Andy Tao, a Purdue biochemist and senior author of the study. Because molecular interactions and protein functions are disturbed when samples are collected, researchers can't obtain an accurate picture of biochemical mechanisms related to illnesses such as cancer and heart disease.
Tao and his research team used dendrimers because they can pass through cell walls efficiently with little disturbance to the cells and then label specific proteins with isotopic tags while cells are still alive. This allows the scientists to determine the activities of proteins that play roles in specific diseases. Proteins carry genetic messages throughout the cell causing biochemical changes that can determine whether a cell behaves normally or abnormally. Proteins also are important in directing immune responses.
The Purdue scientists report on their new strategy to discover proteins and protein levels, called soluble polymer-based isotopic labeling (SoPIL), in the current issue of the journal Chemical Communications. The study also is featured in the journal's news publication Chemical Biology.
"The problem with the current method of using proteomics - protein profiling - is that we use very small sample amounts so sensitive that we can't effectively use existing technologies to study them," Tao said. "In addition, to study a specific protein and its function, we want to preserve its natural environment and see where two molecules meet and what the interaction is when they bind.
"Taking small samples of blood, cells or tissue to study extracted proteins in laboratory dishes damages the sample and the natural environment is destroyed."
The dendrimers would carry one of the stable isotopic or fluorescent labels to identify the presence or absence of a protein that can be further developed for use as a disease indicator, or biomarker.
Snake venom cells were used because they have a very high concentration of proteins similar to some found in human blood, Tao said. The proteins apparently are part of the biochemical process that affects blood clotting or hemorrhage. Understanding how the proteins behave could help determine predisposition to heart disease and cancer and also be useful in diagnosis and drug development.
In future research, Tao plans to investigate how dendrimers are able to enter the cell so easily, what happens to them once they are in the cell and whether there are any long-term effects.
The other researchers involved with this study were Purdue postdoctoral student Minjie Guo and Purdue graduate student Jacob Galan, both in Tao's laboratory.
Purdue University and the National Institutes of Health's National Heart, Lung and Blood Institute provided funding for this study.
Writer: Susan A. Steeves
Source: Andy Tao
Related Web sites:
W. Andy Tao.
Chemical Biology.
Purdue Department of Biochemistry
National Heart, Lung and Blood Institute.
ABSTRACT
A novel quantitative proteomics reagent based on soluble nanopolymers
Minjie Guo, Jacob Galan and W. Andy Tao*
Bi-functionalized dendrimers lead to highly efficient quantitative proteomics and the determination of protease activities in snake venoms. Quantitative proteomics holds significant promise for the discovery of diagnostic or prognostic protein markers and for the detection of new therapeutic targets.
1) The isotope-coded affinity tagging (ICAT) method is perhaps the best characterized approach for quantitative proteomics that combines stable isotope labeling with affinity purification.
2) Among other popular methods developed since then, the adaptation of solid-phase capture and release process is an important improvement for isotope tagging and selective peptide isolation. This procedure is simple and efficient and has the potential for automation and high-throughput experiments.
However, the most notable liability of solid phase capture is the heterogeneous reaction conditions, which can exhibit several of the following problems: nonlinear kinetic behavior, unequal distribution and/or access to chemical reagents, and solvation problems. Due to extreme complexity and proteins in low-abundance, the heterogeneous nature of solid-phase reaction presents a significant issue for proteomic sample recovery. Here we devise a new strategy, termed Soluble Polymer-based Isotopic Labeling (SoPIL).
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