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Originally published In Press as doi:10.1074/mcp.M500145-MCP200 on July 18, 2005.
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Molecular & Cellular Proteomics 4:1602-1613, 2005.
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.


Research

Functional Screening of Serine Protease Inhibitors in the Medical Leech Hirudo medicinalis Monitored by Intensity Fading MALDI-TOF MS*

Oscar Yanes{ddagger}, Josep Villanueva§, Enrique Querol and Francesc X. Aviles

From the Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain

The blood-feeding invertebrates are a rich biological source of drugs and lead compounds to treat cardiovascular diseases because they have evolved highly efficient mechanisms to feed on their hosts by blocking blood coagulation. In this work, we focused our attention on the leech Hirudo medicinalis. We performed, by "intensity fading" MALDI-TOF mass spectrometry, a comprehensive detection and functional analysis of pre-existent peptides and small proteins with the capability of binding to trypsin-like proteases related to blood coagulation. Combining "intensity fading MS" and off-line LC prefractionation allowed us to detect more than 75 molecules present in the leech extract that interact specifically with a trypsin-like protease over a sample profile of nearly 2,000 different peptides/proteins in the 2–20-kDa range. Moreover we resolved 232 individual components from the complex mixture, 13 of which have high sequence homology with previously described serine protease inhibitors. Our findings indicate that such extracts are much more complex than expected. Additionally, intensity fading MS, when complemented with LC separation strategies, seems to be a useful tool to investigate complex biological samples, establishing a new bridge between profiling, functional peptidomics, and subsequent drug discovery.


§ To whom correspondence may be addressed: Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021. Tel.: 212-639-6676; Fax: 212-717-3604; E-mail: villanj1{at}mskcc.org

To whom correspondence may be addressed: Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain. Tel.: 34-93-581-1315; Fax: 34-93-581-2011; E-mail: FrancescXavier.Aviles{at}uab.es


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