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New Technique Promises Mass Spec Analysis of C-terminal Peptides


This story originally ran on June 23.

By Adam Bonislawski

A research team composed of scientists from the Institute of Molecular Medicine and Cell Research of the University of Freiburg and the University of British Columbia has developed a new approach for identifying and analyzing protein carboxy termini using mass spectrometry.

The technique, which the researchers describe in an article in the June issue of Nature Methods, uses high-molecular-weight polymers to enable the enrichment of C-terminal peptides and their subsequent analysis via mass spec. It allows for the first time the proteome-wide study of protein carboxy termini, Oliver Schilling, a researcher at the University of Freiburg and one of the study's authors, told ProteoMonitor.

Because proteolysis is a key process in the alteration of protein function, the study of new protein N and C termini generated through proteolysis offers potentially important insights into physiological pathways and the processes of diseases like cancer. Techniques for proteome-wide analysis of N termini have existed for several years. Due largely to the lower reactivity of carboxyl groups, however, no similar techniques have been available for the study of C termini.

"We looked through the literature, and we couldn't find any approach that had specifically targeted and isolated protein C termini from entire proteins," Schilling said. "There were some approaches that used cation-exchange chromatography where you could see some light enrichment of C termini, but nothing that specifically isolated them."

To enrich C-terminal peptides for analysis by mass spectrometry, the researchers first chemically protected the protein amine and carboxyl groups. They then performed a tryptic digest after which they chemically protected the newly formed amine groups while leaving the newly formed carboxyl groups free. They then coupled these free carboxyl groups to a high-molecular-weight linear polyallylamine polymer by EDC-mediated condensation of the carboxyl groups to the primary amines of the polymer. The original C-terminal peptides with their blocked carboxyl group remained uncoupled, allowing them to be separated from the polymer-bound peptides using ultrafiltration.

"The real novelty is this enrichment strategy," Schilling said. "And a key step is the use of the polymer to react with the internal peptides. When we tried standard solid-phase chemistry, let's say magnetic beads with a primary amine surface, the pullout wasn't very efficient."

Also key to the process was the chemical protection of the proteins' lysine residues, which generated peptides with ArgC-like specificity during tryptic digest, resulting in longer peptide sequences and significantly improved identification rates during mass spec.

After enrichment, the researchers analyzed the C-terminal peptides using LC MS/MS on an AB Sciex QSTAR mass spectrometer. Applying the technique to Escherichia coli lysates, they found that roughly 40 percent of the identified carboxy termini were the results of protein cleavage – a number comparable to the roughly 50 percent of N-terminally processed proteins found in previous studies of the E. coli proteome.

The researchers also developed a modified version of the method that incorporates stable-isotope labeling of C-terminal peptides, which allows for the quantitative comparison of C termini from different samples to distinguish induced proteolysis from background proteolysis.

"So we have control samples and the protease-incubated samples. Or you could have an animal with the protease and an animal lacking the protease – a knockout mouse, for example," Schilling said. "We prep our samples, we use differential isotope labeling, and then we can isolate the C termini, and the C termini that shows up in both samples gives us an idea of the ongoing background proteolysis, whereas something that is only around when we add our protease to both samples is a C terminus generated by the protease under investigation, and that allows us to specifically investigate cleavages of that particular protease."

The technique was designed, Schilling said, to encourage easy adoption by other researchers.

"We made sure this approach uses standard laboratory reagents, so that everything used here can be ordered from any standard lab supplier," he said. "We did this to make sure that it's widely applicable."

Schilling and his team at the Institute of Molecular Medicine and Cell Research are presently using the method to study cathepsin proteases and their roles in tumorigenesis. The technique should enhance the study of a wide variety of processes, he said, allowing scientists to better understand what truncations occur at protein C termini and to investigate how they influence biological processes.

"This sheds light on a part of the protein that wasn't accessible before," he said. "There are more than 560 proteases in humans that are basically involved in every disease process, every pathological process, every physiological process, every developmental process. But for most proteases it's not known exactly what they cleave, and this is where the motivation for this process comes from."

Schilling likened the approach to analyzing phosphorylation sites. "This idea is the same – you have site-specific alterations of proteins. In one case you put a phosphate group on them, and in the other case you make a little truncation."

Other research groups have recently made progress in the proteomic analysis of protein C termini, as well.

In a study, also published this month in Nature Methods, a team of scientists from the University of Ghent and the Autonomous University of Barcelona demonstrated a positional proteomics approach for analyzing C-terminal peptides in human cell lysates.