All day long, cells churn out proteins—cytokines, chemokines, growth factors, and more. Some of these proteins are secreted; that is, they are pushed along a pathway, tumbling through the endoplasmic reticulum, into the Golgi apparatus where they are packaged and processed, and shipped through the cell membrane and into the extracellular space. Many secreted proteins are used to signal other cells. For example, since cancer cells secrete proteins to direct invasion and metastasis, learning what proteins these are can help researchers discover biomarkers for the disease.
secretome has been difficult. That's because scientists have to rely on measuring these proteins after they are secreted into the culture medium in which the cells grow. That culture medium usually includes bovine serum, which is filled with contaminants that are similar to human protein sequences. The cell's secreted proteins are not very abundant compared to the background noise of the serum proteins. "Between the contamination from the cell culture medium and the low abundance of secreted proteins, the signal-to-noise ratio is very low," says North Carolina State University (NCSU) assistant professor Balaji Rao.
To counter this effect, researchers have tried to remove the serum, but this has an undesirable side effect: the cells change. Whenever scientists change the native environment of the cell, the cell responds by altering the proteins it generates, folds, transports and secretes. Without the serum, cellular physiology is altered and a different set of proteins is secreted. And for scientists who study human embryonic stem cells, like Rao, any change in culture conditions can cause the cells to differentiate, further complicating the picture.
Enriching the Signal
If background noise is the problem, why not try to increase the signal-to-noise ratio by capturing the secreted proteins more efficiently? This was the idea behind a recent paper from the lab of Jeroen Krijgsveld, head of the Proteomic Core Facility at the European Molecular Biology Laboratory (EMBL). Katrin Eichelbaum, a postdoc on Krigsveld’s team, and colleagues published a paper in Nature Biotechnology describing a technique that uses an azide-containing amino acid to label newly synthesized proteins and then captures those proteins with a click chemistry reaction (1). To see how well this new protocol works, they combined it with another technique, stable isotope labeling with amino acids in cell culture, called SILAC for short.
SILAC replaces natural amino acids with a stable isotope-labeled version. For example, it might substitute carbon-13 for carbon-12 in particular amino acids. The labeled amino acids are added to the growth medium, taken up by the cells, and subsequently incorporated into newly synthesized proteins. The two versions of the proteins, labeled and not labeled, act the same biologically and can be processed together. But mass spectrometry can be used to quantify the number of proteins from each sample, due to the difference in mass between the isotope-labeled amino acids and the unlabeled ones. This allows researchers to look at the relative quantity of proteins in two different samples.
In the study, Eichelbaum and Krijsveld labeled cells with a pulse of azidohomoalanine (AHA), an azide-bearing analog of methionine, allowing them to selectively capture newly synthesized proteins secreted into the cell medium to an alkyne-activated resin using click chemistry. They also used pulsed SILAC (pSILAC), exposing two parallel cell populations with either intermediate- or heavy-labeled arginine and lysine for a precise period of time. Thus, they were able to quantify the relative amounts of different proteins in two samples: one where they used pSILAC along with pulsed AHA labeling to try to enrich the number of newly synthesized proteins they could capture, and one using pSILAC alone.
"We found roughly twenty secreted proteins in the non-enriched sample, compared with something on the order of six hundred proteins in the enriched sample. We saw a huge improvement," says Krijsveld. "We have found many proteins that are rarely seen in proteomic studies. We are really tapping into a new source of proteins that are important for cell communication and regulation."
Just like any approach that collects proteins that are already in the serum, this technique still contains some contamination from the cell culture medium, but Krijsveld’s team is working on reducing that noise even further. And since AHA is a methionine homolog, it will only be incorporated into proteins containing methionine, which means that about five percent of proteins in mammalian cells get missed with his approach. "We find that acceptable," he says.
The next step is to expand the protocol and apply it to a variety of different cells. Krijsveld is also very interested in protein turnover and distinguishing newly synthesized proteins from old, so he plans to take this approach to study not only the secretome but protein synthesis within the cell as well.
At NCSU, stem cell biologist Rao has been collaborating with David Muddiman, a NCSU professor of chemistry with expertise in mass spectrometry and proteomics for about five years. They had an idea that could turn the study of the secretome on its ear: instead of looking at the secreted proteins once they're outside the cell, why not catch them as they traverse through the secretory organelles? So they developed an isolation protocol that made sure that the organelles didn't rupture during separation, which was described in a paper in Molecular and Cellular Proteomics (2). "It's a centrifugation-based protocol, essentially," says Rao. "It doesn't require any specialized equipment but borrows from other protocols. We tuned it so that we are not rupturing the membranes of the organelles."
While previous protocols for isolating the secretory organelles have focused on getting pure organelles—just endoplasmic reticulum (ER) or just Golgi—Rao and Muddiman’s focus was on the cargo inside the organelles. Once the organelles were separated, the group used mass spectrometry to make a list of proteins found. Next, they compared that list to a database to rule out proteins that are part of the organelles' membranes themselves, leaving only those that are the proteins inside the organelles. Although they did have some contamination from the organelles, Rao says that this procedure could be fine-tuned to increase purity and increase the signal, but they wanted to make sure they didn't miss any possible secreted proteins in this experiment.
There are a few advantages to this approach. “The secretomes of different cell types growing in co-culture experiments can be separately probed using this method,” says Muddiman. That isn’t something that can be done with any other method. And researchers can measure the changes in protein secretion over time for a single cell population. This is because the cells don’t have to be grown in cell culture at all for this technique. In fact, primary tissue—such as tissue donated by patients—can be directly analyzed. "What we get is a snapshot of the proteins produced by the cell during a specific time frame," says Rao. He anticipates next refining the protocol and trying it on other cell types.
Researchers are hopeful that these new methods can help elucidate the role of secreted proteins in the life of the cell, leading to a greater understanding of stem cells, cancer cells, and cell communication. And, the methods can possibly be combined for even greater efficacy. According to Rao, there's no reason why his approach of isolating and purifying the secretory organelles can't be combined with the amino-acid labeling technique used by Eichelbaum and Krijsveld to increase the signal-to-noise ratio still further.
- Eichelbaum, K., M. Winter, M. B. B. Diaz, S. Herzig, and J. Krijgsveld. 2012. Selective enrichment of newly synthesized proteins for quantitative secretome analysis. Nature Biotechnology 30(10):984-990.
- Sarkar, P., S. M. Randall, D. C. Muddiman, and B. M. Rao. 2012. Targeted proteomics of the secretory pathway reveals the secretome of mouse embryonic fibroblasts and human embryonic stem cells. Molecular & Cellular Proteomics 11(12):1829-1839.