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Many cellular activities are mediated by conformational changes in proteins or involve rearrangement of protein assemblies. These motions are now commonly investigated in vitro as well as at the single-molecule level. But Dennis Discher and his colleagues at the University of Pennsylvania wanted to develop an in-cell method to study these motions.

They were interested in studying molecular responses in cells under stress and initally developed their labeling technique in human red blood cells. The premise was to label cysteines with thiol-reactive probes in both stressed and unstressed cells. Then, differential labeling of proteins would indicate that under stress, previously buried cysteine residues became exposed and thus accessible to the fluorescent probe.

To implement this, the researchers reversibly lysed the red blood cells, loaded them with a thiol-reactive fluorophore, resealed them and then sheared a fraction of the cells using a fluid shearing device. After incubation to allow labeling, they lysed the cells again and quenched the unreacted label.

Immediately they could analyze these reactions by SDS-PAGE and also image the cells to see what parts of cells were differentially labeled. Having thus gotten an idea of what to expect, they then excised the bands of interest and subjected them to quantitative mass spectrometry analysis. “So you can run gels, and do a first analysis at the bench without sending every sample to a mass spec,” summarizes Discher. “Mass spectrometry then positively identifies what proteins are labeled, and which cysteines are hit and by how much.”

In the case of red blood cell shear stress, Discher's team found that the structural spectrin proteins were differentially labeled. This provided confirmation that the membrane cytoskeleton is important for this cell type's deformability under the stresses of blood flow. More specifically, they identified the cysteines that were labeled in the sheared cells, pinpointing which domains in spectrin unfold under stress. They also applied this method to study a more complicated system, the mesenchymal stem cell. Analysis of tensed and drug-relaxed cells provided a proteomic short-list of prominent structural proteins and also revealed differential labeling of several cytoskeletal proteins such as nonmuscle myosin and vimentin.

As with any label, the fluorescent probe can alter protein function or be toxic to cells, so the key to applying this technique is to find a toxicity threshold for each experiment. “There might be functions that these probes affect,” cautions Discher, “but in the short term, the labeling of structural proteins is not devastating to the cell.”

Used with caution, cysteines can also be engineered into proteins to query structural perturbations in vivo using this method. “You want to hide the cysteine in the quaternary structure or in a fold, so that it can become exposed,” says Discher. “And that is the real challenge, to do that without perturbing function, but to do that so that hidden cysteines are exposed upon perturbation.”

Recent advances in mass spectrometry made this analysis possible, and future improvements will allow more sensitive detection of probes. Discher points out that this study could not have been done just several years ago, and presently available technologies can detect the cysteine modification on thousands of peptides, especially from large structural proteins.

“The fluorescence gives you instant gratification,” he points out, adding that now researchers “can bring it to the latest modern levels with mass spec.”