New technique for IDing proteins secreted by cells developed

Sep 25, 2012

(Phys.org)—Researchers from North Carolina State University have developed a new technique to identify the proteins secreted by a cell. The new approach should help researchers collect precise data on cell biology, which is critical in fields ranging from zoology to cancer research.

The work is important because cells communicate by secreting proteins. Some of the proteins act on the cell itself, telling it to grow or multiply, for example. But the proteins can also interact with other cells, influencing them to perform any .

Traditionally, scientists who wanted to identify these proteins and then used mass spectrometry to determine which proteins appeared in the medium the cell was grown on. This has drawbacks, because the proteins of interest are fairly rare compared to the proteins that are already in the medium – which are used to grow and support the cells in the first place. Further, any attempts to culture the cells without these background, supporting proteins affects – skewing the sample.

The new approach takes advantage of the fact that each cell "packages" its proteins in its "secretory pathway." Each cell synthesizes the protein and passes it through this pathway, essentially placing it in a bag-like membrane before it is passed out of the cell.

In their new technique, researchers take a sample of cells and isolate the secretory pathway organelles, which contain the proteins. The researchers then use to analyze the contents of the , in order to see which proteins were being secreted by the cell. Using this approach, the researchers were able to identify proteins that are secreted by human .

"This gives us a snapshot of exactly what a cell was secreting at that point in time," says Dr. Balaji Rao, an assistant professor of chemical and biomolecular engineering at NC State and co-author of a paper describing the work.

This new method eliminates the problems related to the proteins found in cell culture media. But it also allows researchers to track changes in the proteins released by a cell in response to a stimulus, such as exposure to a chemical. This can be done by taking samples at various points in time after cells have been exposed to the stimulus.

And, in principle, this technique would also allow researchers to identify which proteins any specific type of cell is secreting when in a mixed population of cells.

"As long as you can separate the cells you are interested in, this should be possible," says Rao. "And that is important, because most tissues are made up of heterogeneous populations of cells – and communication between those cells is biologically significant."

Explore further: Research sheds light on what causes cells to divide

More information: The paper, "Targeted proteomics of the secretory pathway reveals the secretome of mouse embryonic fibroblasts and human embryonic stem cells," was published online Sept. 15 in the journal Molecular & Cellular Proteomics. www.mcponline.org/content/earl… 9/15/mcp.M112.020503

Abstract:
Proteins endogenously secreted by human embryonic stem cells (hESCs) and those present in hESC culture medium are critical regulators of hESC self-renewal and differentiation. Current MS-based approaches for identifying secreted proteins rely predominantly on MS analysis of cell culture supernatants. Here we show that targeted proteomics of secretory pathway organelles is a powerful alternate approach to interrogate the cellular secretome. We have developed procedures to obtain subcellular fractions from mouse embryonic fibroblasts (MEFs) and hESCs that are enriched in secretory pathway organelles, while ensuring retention of the secretory cargo. MS analysis of these fractions from hESCs cultured in MEF conditioned medium (MEF-CM) or MEFs exposed to hESC medium revealed 99 and 129 proteins putatively secreted by hESCs and MEFs, respectively. Of these, 53 and 62 proteins have been previously identified in cell culture supernatants of MEFs and hESCs respectively, thus establishing the validity of our approach. Furthermore, 76 and 37 putatively secreted proteins identified in this study, in MEFs and hESCs respectively, have not been reported in previous MS analyses. Identification of low abundance secreted proteins by MS analysis of cell culture supernatants typically necessitates the use of altered culture conditions such as serum-free medium. However, an altered medium formulation might directly influence the cellular secretome. Indeed, we observed significant differences between the abundances of several secreted proteins in subcellular fractions isolated from hESCs cultured in MEF-CM and those exposed to unconditioned hESC medium for 24 hours. By contrast, targeted proteomics of secretory pathway organelles does not require the use of customized media. We expect that our approach will be particularly valuable in two contexts highly relevant to hESC biology – to obtain a temporal snapshot of proteins secreted in response to a differentiation trigger, and to identify proteins secreted by cells that are isolated from a heterogeneous population.

Related Stories

MIT probe may help untangle cells' signaling pathways

Jun 27, 2008

MIT researchers have designed a new type of probe that can image thousands of interactions between proteins inside a living cell, giving them a tool to untangle the web of signaling pathways that control most of a cell's ...

Yale scientists map cell signaling network

Nov 30, 2005

Yale University scientists have mapped, for the first time, the proteins and kinase signaling network that control how cells of higher organisms operate.

Researchers discover key to cell specialization

Nov 10, 2011

Researchers at then Albert Einstein College of Medicine of Yeshiva University have uncovered a mechanism that governs how cells become specialized during development. Their findings could have implications for human health ...

Recommended for you

Research sheds light on what causes cells to divide

Dec 24, 2014

When a rapidly-growing cell divides into two smaller cells, what triggers the split? Is it the size the growing cell eventually reaches? Or is the real trigger the time period over which the cell keeps growing ...

Locking mechanism found for 'scissors' that cut DNA

Dec 24, 2014

Researchers at Johns Hopkins have discovered what keeps an enzyme from becoming overzealous in its clipping of DNA. Since controlled clipping is required for the production of specialized immune system proteins, ...

Scrapie could breach the species barrier

Dec 24, 2014

INRA scientists have shown for the first time that the pathogens responsible for scrapie in small ruminants (prions) have the potential to convert the human prion protein from a healthy state to a pathological ...

Extracting bioactive compounds from marine microalgae

Dec 24, 2014

Microalgae can produce high value health compounds like omega-3s , traditionally sourced from fish. With declining fish stocks, an alternative source is imperative. Published in the Pertanika Journal of Tr ...

User comments : 0

Please sign in to add a comment. Registration is free, and takes less than a minute. Read more

Click here to reset your password.
Sign in to get notified via email when new comments are made.