New research from scientists at the University of Birmingham and other institutions describes a method of counting labeled proteins in living cells dubbed Protein-tag Degree of Labeling (PRoDOL) that could be a valuable tool in protein quantification studies. Details of the method are provided in a new Nature Methods paper titled, “ProDOL: a general method to determine the degree of labeling for staining optimization and molecular counting.”
According to the team, the technique offers a robust approach for precisely determining labeling efficiencies, the number of proteins labeled with fluorescent markers in living cells, in microscopy images. They claim that it addresses existing challenges with analyzing protein data from microscopy images. Currently, the accuracy of these analyses depends on being able to determine the degree of labeling or how many proteins are labeled with fluorescent markers.
As they noted in the paper, methods such as “immunolabeling result in variable labeling efficiencies that are difficult to characterize.” Also, while an approach like “genetic fusion with fluorescent proteins can yield a one-to-one ratio of label to the [protein of interest], it is often not suited for quantitative measurements and can be challenging due to insufficient photostability and ill-defined brightness states.”
For its part, ProDOL uses a “modular DOL calibration probe that employs a fluorescent protein as a nearly background-free reference signal combined with protein tags,” the research team wrote. “This construct can be transiently or stably expressed in various cell lines and provides a way to measure labeling efficiency through colocalization at the single-molecule level, thus enhancing the reliability and versatility of the measurements.” They noted that the calibration probe is made up of “a membrane-anchored enhanced green fluorescent protein (eGFP) fused to a SNAP-tag and HaloTag” although other protein tags could be used as well. The scientists also designed an analysis pipeline specifically for performing “labeling efficiency measurements by single-molecule colocalization analysis.”
The paper includes an example of the kinds of insights that ProDOL can provide. Specifically, the scientists used it to investigate how an HIV-1 pathogenesis factor called Nef affects the response of CD4 T immune cells. They used ProDOL to count both the total and activated copy numbers of the adapter protein SLP-76 in tiny signaling clusters of immune cells. They demonstrated that for SLP-76, “the viral protein primarily acts to prevent recruitment of host cell components into signaling hubs” among other findings.
Dirk-Peter Herten, PhD, lead researcher on the study and chair in cell biology of membrane proteins at the University of Birmingham believes that the tool could “become a standard and valuable tool in the field of biomedical research, paving the way for more precise and insightful studies of cellular functions.” It could, for example, “improve our understanding of cellular signaling processes in the activation of immune cells or platelets which are relevant in the context of various diseases, including but not limited to inflammation, immune deficiencies, and various cardiovascular diseases,” Herten said. It is also highly versatile, making it applicable to various cell types and experimental conditions.
