A technology that allows scientists to isolate and interrogate a cell’s secretory pathway organelles has demonstrated that current techniques for analyzing a cell’s secretory output may be missing a large number of potentially key proteins. One way that cells communicate with each other is through the production and signalling of secretory proteins, and current techniques for identifying such proteins uses mass spectrometry-based analysis of the growth medium to see what the cells are adding to what’s already present.
However, due to the mix of proteins and compounds contained in the growth medium to support the cultured cells, this method isn’t ideal. Furthermore, using MS to identify low-abundance secreted proteins typically requires the use of altered culture conditions such as serum-free medium, which can itself impact on the cell’s secretory output.
Researchers at North Carolina State University (NC State) have now developed a platform that they claim allows them to isolate secretory pathway organelles from cells and analyze the contents of the intact organelles by mass spectrometry directly. “This gives us a snapshot of exactly what a cell was secreting at that point in time,” explains Balaji Rao, who heads the team at NC State.
The researchers used the technique to isolate the subcellular secretory pathway fractions from mouse embryonic fibroblasts (MEFs) and human embryonic stem cells (hESCs). MS analysis of the fractions identified 99 proteins putatively secreted by the MEFs, and 129 by the hESCs. Importantly, of these, 76 MEF proteins and 37 hESC proteins hadn’t previously been reported in MS analyses.
A major effect of the medium formulation on the proteins secreted by cells was also observed. There were significant differences between the abundances of several secreted proteins produced by hESCs cultured in MEF-CM, and those exposed to unconditional hESC medium.
The new technique could feasibly allow researchers to monitor a cell’s secretory output in response to a range of applied stimuli and chemicals, and identify proteins that are secreted by one cell type in a heterogeneous cell population. “As long as you can separate the cells you are interested in, this should be possible,” Dr. Rao claims. “And that is important, because most tissues are made up of heterogeneous populations of cells—and communication between those cells is biologically significant.”
Dr. Rao et al., describe their technology and its application in Molecular & Cellular Proteomics, in a paper titled “Targeted proteomics of the secretory pathway reveals the secretome of mouse embryonic fibroblasts and human embryonic stem cells.”