Analytical Trick Improves Efficiency of Protein Analysis

Chemists have recently begun to double-up with two different samples in a single run, e.g., to compare normal to diseased tissue to examine how the proteins change. Now, researchers at the University of Wisconsin-Madison (WISC; Madison, WI; USA) led by Joshua Coon, professor of chemistry and biomolecular chemistry, have built on this by developing a technique, termed “neutron-encoding,” with potential to multiplex 20 samples at once.

In the current study, the team investigated mouse tissue for potential biochemical pathways underlying the correlation between caloric restriction and extended lifespan previously observed in many animals. "Some of these mice have lost a certain gene related to metabolism, so we are comparing four types of tissue all at once. We can look at the brain, liver, or heart, and ask how does the abundance of proteins vary?" said first author and graduate student Alexander Hebert.

The team has performed six simultaneous analyses using the new technique; it could do batches of 20. Key to the existing, original doubling-up technique was synthetically inserting stable isotope tags into the amino acids used in metabolic labeling of proteins. In preparing two samples, one sample would receive an amino acid containing common isotopes, the other containing heavier isotopes. The result—chemically identical proteins with slightly different masses easily identified by MS.

The new report describes the use of amino acids built from a broader range of isotopes that would be expected to have identical mass, but do not since part of their mass has been converted to energy required to hold the atomic nuclei together. Without this energy, the positively charged proteins would repel each other and the atomic nucleus would be destroyed. The tiny loss of mass due to this conversion to binding energy can be detected in the new, ultra-precise mass spectrometers. The mass difference in the new technique is more than 1,000 times below the mass differences in the existing doubled-up technique, but it is enough to quantify and identify proteins from at least 6, and theoretically, 20 samples at once.

Prof. Coon added, "We could look for protein differences in cells from 100 different tumors. The proteins might reveal that you are dealing with 5 or 10 distinct syndromes in this seemingly identical cancer, which could suggest treatments that are more tailored to the individual. If you compare proteins in normal versus tumor tissue, you might find a certain protein at uncommonly high concentrations, or [that] was modified in certain ways. You might identify a protein that would help diagnose this cancer sooner [or] a protein that is so vital to the cancer that it would make an ideal target for a new drug."

The new procedure was described advanced online February 24, 2013, in the journal Nature Methods.

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