Detecting picograms of protein in the secretome

Posted by Kasra

When designing experiments in the lab, we usually say we cannot check for everything. Well, what if we could?! Meissner et al. used only 150,000 macrophages per sample to analyze their secretome. They have been able to detect and quantify protein abundances at the picogram level in a label-free system. Picogram detection limit means that cytokines are readily quantifiable, and Meisner et al. claim it matches with the detection limit of ELISA. I could imagine that in not so long this technology can become more readily accessible, allowing researchers to acquire more pertinent data per sample. We can know the statuses of a multitude of proteins (from cytokines to nonconventionally secreted proteins) simultaneously, not through multiple ELISAs and western blots, and therefore draw much more sensible conclusions from experiments. It will actually be more affordable than separate quantification of each protein, not mentioning dramatically time-saving.

Also, this data can be integrated with other high throughput quantitative analyses of the cell. For instance, Meissner et al. have compared the changes in protein abundance in the secretome to changes in their transcript levels, and roles of different adaptor molecules (in this case MyD88 versus TRIF) and tried to explain how they all relate.

Meissner F, Scheltema RA, Mollenkopf HJ, & Mann M (2013). Direct proteomic quantification of the secretome of activated immune cells. Science (New York, N.Y.), 340 (6131), 475-8 PMID: 23620052

ResearchBlogging.org

Studying the secretome of Leishmania donovani

Posted by Kasra Hassani

In this paper, Silverman et al. have pointed to two interesting subjects: first, what proteins are generally secreted from Leishmania, and second, how are these proteins secreted. In an extensive proteomic analysis, they have pointed out 151 proteins that they believe are being actively secreted out of stationary promastigotes of Leishmania donovani. These proteins belong to a wide variety of groups, such as proteases, antioxidants, nucleases etc. and each might play roles in survival of the parasite within its hosts and modulation of the immune response. Identification of these proteins opens up many opportunities for further studies that promote understanding their function and possible therapeutic targets in continuing studies.

Another interesting finding of Silverman et al. was that among these secreted proteins only 2 contain a classical amino-terminal secretion signal, which means that Leishmania largely might benefit from non-classical secretion pathways such as exosomes. Exosomes have been studied previously in human B cells and dendritic cells and it is actually interesting to point out that there is striking correspondence between the proteome content of these exosomes and Leishmania’s secretome (except for the proteins for which Leishmania does not have an ortholog). The authors have proposed the release of exosomes from the surface of the cell and especially from the flagellar pocket to be an important pathway of protein secretion by Leishmania and they have observed vesicular budding from the parasite surface by STM.