A group of researchers in Spain have used a novel mass spectrometry approach called Intensity Fading MALDI MS to discover small proteins and peptides in leeches that could potentially lead to a treatment for cardiovascular disease by blocking blood coagulation.
"Most heart attacks and strokes are associated with a blocked artery," explained Oscar Yanes, the first author of the study, which was published in this month's issue of Molecular and Cellular Protoemics. "In some cases, blood clots may cause the blockage of arteries that leads to cardiovascular disease. People with cardiovascular disease typically have an increased tendency to form blood clots, and a decreased ability to dissolve clots before they can do any damage. Therefore, compounds interrupting the blood coagulation cascade may inhibit thrombus development."
Originally developed by Josep Villanueva at the Universitat Autonoma de Barcelona, the Intensity Fading MALDI MS technique relies on a decrease in mass spec signal intensity to indicate that a molecule has bound to a ligand. The method is useful for screening complex mixtures in a high-throughput fashion to see if there are molecules within a mixture that bind to the ligand.
"By doing MALDI you can screen a huge amount of compounds, then go on to second-round techniques if you want to calculate the binding coefficient," said Villanueva, who is now a research associate at the Memorial Sloan-Kettering Cancer Center in New York City.
"By doing MALDI you can screen a huge amount of compounds, then go on to second round techniques if you want to calculate the binding coefficient."
To perform the technique, researchers mix together the compound mixture with the ligand of interest in conditions where the proteins would normally bind. They then combine that mixture with the MALDI matrix compound that helps in the ionization process. The sample is then loaded onto a MALDI mass spectrometer to screen for any decreases in intensity as compared to the MALDI mass-spec profile of the unbound mixture.
"Sometimes the signal intensity goes down by 70, 80 percent," said Villanueva.
The Intensity Fading MALDI MS technique was first discovered serendipitously when researchers were trying to look for a signal in the mass spec that would represent a target bound to a ligand, Villanueva said. Instead, researchers found that the signal intensity of both the target and the ligand went down.
"If a target had an m/z of 20,000, and a ligand 1,000, we were trying to see the 21,000 signal in the mass spec," Villanueva said. "We couldn't, and at the same time we saw that the intensity of the proper ligand would go down. The m/z range was disturbed."
With the leech study, Villanueva and his colleagues chose serine protease as the ligand, and screened extract from the salivary glands of the leech Hirudo medicinalis for molecules that would bind to the ligand. Most coagulation factors involved in the blood coagulation cascade are enzymes that belong to the serine protease family, so in theory, molecules that can bind to and block the action of serine proteases should be able to prevent coagulation.
Researchers screened nearly 2,000 proteins and peptides in the leech extract. Of those, more than 75 interacted specifically with serine protease. The researchers then purified those proteins and peptides by chromatography, and tried to sequence them using the Edmund degradation technique.
With some of the proteins and peptides, Edmund degradation was not possible because the N-terminal was blocked. In the end, the researchers ended up with 17 proteins and peptides that they could purify and sequence.
Finally, the researchers used classical enzymology assays to test if the 17 proteins and peptides did in fact inhibit the activity of serine protease. Of the 17, 16 inhibited the enzyme, while one did not.
The next step in taking the 16 molecules closer towards therapy would be to further characterize the activity of each of the molecules, Villanueva said.
"There are different enzymes in the [coagulation] cascade, and we don't know how specific these inhibitors are for the key enzymes in the coagulation. The next step would be to take all these key enzymes and to test the inhibitors on them," he added.
Villanueva noted that this is the first time that the Intensity Fading MALDI MS technique has been shown to work on a real biological extract. For the original proof-of-principle study, which was published in 2003 in Analytical Chemistry, researchers prepared artificial mixtures and then screened for binding.
"This is the first real test," said Villanueva.
Yanes said that he and his colleagues at the Universitat Autonoma de Barcelona are going to continue to apply their approach to screening serine protease binding to other types of leeches, as well as different blood-sucking animals, in the hopes of expanding their repertoire of serine protease blockers.
"Leeches belong to an extensive family with a large number of species and subspecies which have evolved highly efficient mechanisms to block blood coagulation," said Yanes. "Considering the fast adaptation to this kind of nutrition, resulting in accelerated evolutionary selection and fixation of very specific interactions between serine protease inhibitors of the leech and proteases of the host, we think that screening additional types of leeches will lead to the discovery of very specific compounds blocking specific serine proteases of the coagulation cascade."
Other Barcelona researchers involved with the leech study were Francesc Aviles and Enrique Querol.
Tien-Shun Lee ([email protected])