Aebersold s Group Develops New Method To Identify, Quantify Phosphorylation Sites

Researchers led by Ruedi Aebersold said they have come up with a way to identify and relatively quantify peptide phophorylation sites with greater speed and robustness than previously possible by enriching phosphorylated peptides within complex protein mixtures prior to analysis by mass spectrometry.

The research, which is believed to be the first to suggest that synthetic polyamine molecules could be effective in a proteomics application, opens a window for proteomic researchers to find new ways to deal with biochemical problems in the polymer and organic chemistry fields.

According to Aebersold, whose team at the University of Zurich led the study with W. Andy Tao at Purdue University, "when you digest a mixture, there are relatively few phosphorylated sites compared to non-phosphorylated sites, so if you just inject a tryptic digest of a protein mixture, it's relatively rare that you hit on a phosphorylated site. What we've tried to do is to selectively isolate the phosphorylated peptides out of a complex peptide mixture."

The key to isolating the phophorylated peptides is a synthetic polyamine molecule called a dendrimer. Tao, an assistant professor of biochemistry at Purdue who is the first author on a paper describing the new method published in the August issue of Nature Methods, chose to use dendrimers because they have a very high density of reactive amino groups.

Tao began working on the phosphopeptide project as a postdoc in Aebersold's laboratory at the Institute for Systems Biology in Seattle. Aebersold recently moved to Zurich, but maintains strong ties at the ISB, which he co-founded in 2000.

Previously, another postdoc in Aebersold's lab had developed his own method for enriching phosphopeptides. However, that method involved seven steps and was relatively complicated, so it never gained much popularity after it was published in 2001, Aebersold said.

Using dendrimers, Tao and his colleagues developed a way to enrich for phosphopeptides in "a single pop." The phosphopeptides covalently couple to the dendrimers in a single step, allowing for recovery via acid hydrolysis and subsequent characterization via LC-MS/MS.

"The dendrimer made [the phosphopeptide enrichment reaction] much more efficient. It's very simple now. Even a non-chemist is going to easily be able to perform the reaction," Aebersold told ProteoMonitor this week.

The new dendrimer-dependent reaction is in the advanced stages of the patent process, Aebersold said. The dendrimer is commercially available, and researchers who read the Nature Methods paper should be able to assemble the rest of the reagents without too much trouble, he added. A kit for phosphopeptide enrichment might be assembled in the future without much trouble, if researchers want it, he said.

Eric Peters, a scientist at the Genomics Institute of the Novartis Research Foundation, wrote in an accompanying News and Views commentary on Tao's new method that the dendrimer work is valuable because it can be applied to phosphorylation profiling, and also because it applies techniques from other, nonbiologically oriented fields to solve a biochemical problem.

"The use of solution polymers, such as dendrimers, as auxiliaries in organic reactions has a long-established history, but this is the first report of their effective use in a proteomics application," Peters wrote. "By looking to the fields of polymer and organic chemistry, proteomic researchers may find new solutions for the complex biochemical problems they face."

To demonstrate that the new technique works, Aebersold's team used the method to study changes in protein phosphorylation in lymphocytes after the cells were stimulated with a phosphotase inhibitor. The researchers found all previously known tyrosine phosphorylation sites within the immunoreceptor tyrosine-based activation motifs of the T-cell receptor CD3 chains, as well as numerous previously unknown phosphorylation sites.

Aebersold said in the future, his research team may try to apply dendrimers to selectively isolate other types of modified peptides as well, such as glycopeptides, peptides containing sulfide residues, or peptides containing a carboxyl group.

"We think there are many other applications for this dendrimer in proteomics," said Aebersold. "Slight variants of this dendrimer could be used to selectively isolate peptides with any number of functionalities you want to put in."

Currently, the phosphopeptide enrichement procedure takes a day to perform, Aebersold said. Many reactions can be done in parallel, but there are no real shortcuts for doing one quick-and-dirty isolation, he added.

Aebersold's group is currently working on adapting the technique to an automated platform with robots. "A robot can do it," said Aebersold.

Aebersold's group may apply the technique next to studying protein phosphorylation in Drosophila.

"We have a project studying the genes and corresponding proteins that have to do with controlling the size of the cell in flies," Aebersold explained. "It involves a lot of protein phosphorylation, so that's one application we're working on now."

Tao said the biggest challenges in developing the new phosphopeptide enrichment technique were choosing the dendrimer as the polymer, and filtering out the dendrimer once the enrichment step was completed.

"I tried three or four different solid phase polymers before I started working with dendrimers, which are liquid-based polymers," said Tao. "I spent quite a lot of time trying to work out a solid phase method, but the kinetics were very slow."

Aebersold said the challenge for this type of a technique is not the development of each step, but rather getting the whole process optimized and working robustly at a high yield.

"The challenge in this kind of thing is always in the details," said Aebersold. "If each step works, it doesn't mean the whole thing is going to work."

— Tien-Shun Lee ([email protected])