Strategy Devised to Separate and Identify Thousands of Protein Molecules

The human genome has been mapped. Now, it is the proteins turn, a much more challenging job. There are 20,300 genes, but there are millions of distinctive protein molecules in humans, many of which hold answers to questions about disease and targeting treatment.

A team led by Northwestern University (Evanston, IL, USA) chemical biologist Dr. Neil Kelleher has developed a new “top-down” strategy that can rapidly separate and identify thousands of protein molecules. Many have been unconvinced that such an approach, where each protein is studied complete instead of in smaller pieces, could be conducted on such a large level.

The potential of a top-down approach is that the molecular data scientists do gather will be more closely linked to disease. “Accurate identification of proteins could lead to the identification of biomarkers and early detection of disease as well as the ability to track the outcome of treatment,” Dr. Kelleher said. “We are dramatically changing the strategy for understanding protein molecules at the most basic level. This is necessary for the Human Proteome Project -- the mapping of all healthy human proteins in tissues and organs--to really take off.”

Dr. Kelleher reported that his approach is theoretically simple. “We take proteins--those swimming around in cells--and we measure them,” he said. “We weigh proteins precisely and identify them directly. The way everyone else is doing it is by digesting the proteins, cutting them up into smaller bits called peptides, and putting them back together again. I call it the Humpty Dumpty problem.”

The new strategy, according to Dr. Kelleher, solves the “protein isoform problem” of the “bottom-up” approach where the smaller peptides frequently do not map precisely to single human genes. The study was published October 30, 2011, in the journal Nature.

The top-down method can accurately identify which gene produced which protein. The bottom-up technique is only 60%-90% accurate in identifying proteins precisely. “We need to define all the protein molecules in the human body,” Dr. Kelleher said. “First, we need a map of healthy protein forms, which will become a highly valuable reference list for understanding damaged and diseased forms of proteins. Our technology should allow us to get farther down this road faster.”

In the first large-scale demonstration of the top-down method, the researchers were able to identify more than 3,000 protein forms created from 1,043 genes from human HeLa cells. Their goal was to identify which gene each protein comes from--to provide a one-to-one image. They were able to produce this accurate map of thousands of proteins in just a few months. The researchers also can produce the complete atomic composition for each protein. “If a proton is missing, we know about it,” Dr. Kelleher said.

One gene they examined, the HMGA1 gene associated with premature aging of cells, generate approximately 20 different protein forms. Dr. Kelleher’s team developed a four-dimensional separation system that uses separations and mass spectrometry to measure the charge, mass, and weight of each protein as well as how “greasy” a protein is. The software the researchers developed to study the data during years of work prior to the study proved critical to the success of the top-down method. “If you want to know how the proteins in cancer really work and change, top-down mass spectrometry is getting to the point where it can be part of the discussion,” Dr. Kelleher concluded.

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