Technique Analyzes Effect on Proteins of Stress in Normal and Mutated Red Blood Cells

Researchers at the University of Pennsylvania are studying how physical stress affects proteins within red blood cells that spend their lifetimes being pumped and bumped through millions of vessels. They’re using a technique called cysteine shotgun-mass spectrometry to notice how the amino acid cysteine, and proteins containing it, is changing its properties.

“Red blood cells are disks, and they have proteins right below the membrane that give it resilience, like a car tire,” Discher [Dennis Discher, professor of chemical and biomolecular engineering and bioengineering] said. “The cells are filled with hemoglobin like the tires are filled with air, but where the rubber meets the road is the exterior.”
To measure stress in that membrane on an atomic level, the Discher team needed a way to track changes to the shape of those supporting proteins. They found an ideal proxy for that stress in the amino acid cysteine.
Proteins are long chain of amino acids that are tightly folded in on themselves. The order and chemical properties of the acids determine the locations of the folds, which in turn determine the function of the protein. Cysteine is “hydrophobic”; it interacts poorly with water and so it is usually on the inside of a protein. And because stress changes the shape of these folded proteins, Discher reasoned that measuring the degree to which cysteine is exposed would in effect measure how stressed the protein and cells containing it are.
Discher’s team simulated the shear forces originating from the beating heart, which forcefully pumps blood and ultimately pulls apart the folds that keep cysteine on the inside of proteins at the red blood cell membrane, allowing it to bind with a fluorescent marker dye. The team could visually confirm that more stressed cells were more fluorescent under the microscope but actually tested the levels of marked cysteine using mass spectrometry.
“Just like a polymer engineer designing a tire, we’re looking at the relationship between the chemical makeup and the physical stability of the structure and how it performs,” Discher said. “We can use this technique to look at the relationship between structure, flexibility and function.”

Press release: Penn Researchers Develop Technique for Measuring Stressed Molecules in Cells…
Abstract in PNAS: Cysteine shotgun-mass spectrometry (CS-MS) reveals dynamic sequence of protein structure changes within mutant and stressed cells