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EMBL Team Combines Click Chemistry and SILAC Mass Spec for Improved Secretome Analysis


A team led by researchers at the European Molecular Biology Laboratory has developed a new method for quantitative measurement of the secreted proteome, or secretome.

Detailed in a paper published last week in Nature Biotechnology, the technique combines click chemistry with SILAC mass spectrometry to overcome limitations inherent in conventional secretome analyses, Jeroen Krijgsveld, an EMBL researcher and author on the paper, told ProteoMonitor.

Secreted proteins are key components of cellular signaling and a potentially rich source of disease biomarkers and, as such, proteomics researchers have sought to profile them, typically by analyzing culture medium from cell types of interest.

The low abundance of many secreted proteins, however, makes them difficult targets for this sort of analysis due to the complexity of the serum-containing media in which the cells of interest are usually grown. Reducing this complexity enough to detect low-abundance secreted proteins via mass spec requires extensive time-consuming fractionation.

Alternately, researchers doing secretome profiling sometimes choose to grow their cells in serum-free media, thereby eliminating the background issue. However, this subjects the cells to stress conditions, which can alter their activity and the proteins they are secreting, Krijgsveld said.

To get around these limitations, the EMBL team devised an enrichment strategy to enable detection of low-abundance proteins against the background of serum-based media, labeling proteins with azidohomoalanine, or AHA, an azide-containing analog of the amino acid methionine. The azide-labeled proteins could then be captured by alkyne-activated beads via click chemistry, allowing for pull-down of secreted proteins prior to mass spec analysis.

They combined this click chemistry-based enrichment with pulsed SILAC – exposing two sets of cells to "medium" or "heavy" labeled amino acids for a window of time – to enable quantitative comparisons of secretome contents.

In an analysis of PC3 cells – prostatic adenocarcinoma cells initiated from a bone metastasis — and WPMY-1 cells – myofibroblast stromal cells from healthy prostate – the researchers identified a total of 1,136 proteins, reliably quantifying 684, of which 601 were differentially expressed between the two lines.

They followed this with a comparison of mouse hepatic cells, comparing the hepatoma cell lines Hepa1-6 and Hepa1c1 to primary hepatocytes, finding as many as 581 differentially secreted proteins between them.

The researchers also used the technique to analyze time-resolved secretome changes, investigating the responses at different times of macrophages stimulated by lipopolysaccharides and identifying 97 proteins differentially secreted in at least one of three time points.

Such time-resolved analyses could provide significant mechanistic insights into cell behavior, Krijgsveld said, noting that this was a primary interest of his group.

"The direction we're looking is more a mechanistic point of view to see how the secretome composition can explain the activation of cells and what mechanisms may be underlying [this activation]," he said. "If you think about immune cells, for instance, it's their job to signal infection and damage to tissues and organs, and secreted proteins are key components of [that] signaling function."

"We did this in the paper with the macrophages, and you see a really robust change in the secretome when you stimulate them," he added.

The search for disease biomarkers is also "one of the motivations for looking into this problem," Krijgsveld said, although he added that this was not a primary area of focus for his group.

They are, however, investigating secretome changes as biomarkers for drug response, he said.

"We are focusing on cancer cells and what I think is an interesting idea looking at cancer cells in combination with drug treatment," Krijgsveld said. "In particular [we are] looking to see if there are any changes in the secretome [that we can use] as an indicator for drug sensitivity or resistance in the cells."

For this work, the EMBL team is collaborating with the German Cancer Research Center, which also participated in the Nature Biotechnology study. Currently, Krijgsveld said, the researchers are characterizing large panels of untreated cells to establish profiles of their secretomes at basal levels.

Having quantified just under 700 proteins, the researchers have measured around a quarter of the 2,000 to 2,500 proteins predicted to compose a typical mammalian secretome. Given the high level of specificity they found among cell secretomes, adding new cells types to their analysis will be key to achieving broader coverage, Krijgsveld suggested.

"Although we saw some overlap between cell types, many of the proteins were quite specific to the cells we studied," he said. "So what we learned is that the secretome is a pretty accurate signature of the cell under investigation… a pretty good indication of cell identity or cell status."

"With every cell type we added to the study, we identified new proteins," he added. "So I think if we expanded the number of cells we would get a higher number – closer to 2,000, 2,500."

Improvements in the technique could also expand its reach, Krijgsveld said, noting that, in particular, the researchers are looking into altering the click chemistry in ways that could further reduce background.

One limitation, he noted, is the fact that the method is currently only applicable to methionine, which has a tRNA flexible enough that it can be manipulated to incorporate AHA.

"If there was something for [replacing] leucine or lysine, very abundant amino acids, that would be great, because every protein has them," Krijgsveld said. "But as far as I know, that doesn't exist."

The researchers looked into patenting the technique, but "all of the methods that we used have been used before and all the components we used are commercially available," he said.