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

Abstract

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 trypsinlike 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. Molecular & Cellular Proteomics 4:1602-1613, 2005.Although the task of identifying and characterizing genes in many sequenced genomes in the last years has been very intense, it is even more challenging when applied to the much higher complex field of the protein world (1). One difficult set of proteins for such analysis are those which we could term as "small" (i.e. below 15-20 kDa) (2) because of their variety and difficulty to be detected by several established proteomic approaches such as 2D 1 electrophoresis (3) or computational genomic scanning (4) among others. Therefore, the development of efficient and high throughput technologies for producing functionally related data of peptides and small proteins (2-20 kDa) is increasingly attracting attention. Some of these proteins have potential use for diagnostics or therapeutics (5-7). They include families of peptide hormones, neuropeptides, cytokines, growth factors, and enzyme inhibitors acting as biochemical messengers or modulators that organize the regulatory processes in all organisms (6). In fact, most of the important biopharmaceutical products approved for therapeutic applications are molecules within a molecular mass range of 1-40 kDa (8).The proteomic strategy focused on the analysis of peptides and small proteins from complex mixtures has been named "peptidomics" (9, 10). The primary proteomic tool, 2D gel electrophoresis with mass spectrometry, has been gradually enhanced, and sometimes replaced, by three main technologies: MALDI MS in combination with LC (off line) (9, 11, 16), ESI MS combined with nano-LC (on line) (12-16), and MALDI-TOF MS for in situ peptide profiling (17-21).One of the main goals of these technologies is to provide a compre...

Exploring the “intensity fading” phenomenon in the study of noncovalent interactions by MALDI-TOF mass spectrometry

“…Even within the soft ionization techniques, ESI has been long recognized as the method of choice for studying noncovalenté complexesé [21].é However,é toé date,é we strongly believe that the real potential of MALDI mass spectrometry in the study of noncovalent interactions has been underestimated because of our limitation of a complete understanding of all processes underlying this technology. New experimental approaches not conceived until recent years like "intensity fading MS" or possibleémodificationsébasedéonétheésameéprincipleé [28,36],éinfraredé(IR)éMALDIé [38,é39],éatmosphericépressure (AP)éMALDIé [40,é41],éDIOS-MSé [42-é 44],éorénewégener-ationéoféionédetectorsé [45,é46],émayéputéonétheésameélevel MALDI and ESI mass spectrometry in the field of noncovalent interactions.…”

Identification of Tight-Binding Plasmepsin II and Falcipain 2 Inhibitors in Aqueous Extracts of Marine Invertebrates by the Combination of Enzymatic and Interaction-Based Assays

“…Protease-inhibitor interactions are detected through the decrease (fading) of the relative intensities of the m / z signal corresponding to the inhibitor after the addition of the target protease immobilized to an appropriate support. To confirm the binding, the formed complexes are then dissociated to regenerate the faded ion signal corresponding to the inhibitor [51,52]. Analysis of the positive P. homomalla (heat) extract confirmed the existence of several molecules interacting specifically with Sepharose-immobilized Papain (Figure 6A), corresponding to m / z + of 5973.8, 6074.2, 7357.7, 14711.0 and 14736.9 [53].…”

“…during screening of crude extracts. In contrast, interaction-based assays, such as affinity chromatography [46,49], Surface Plasmon Resonance (SPR)-biosensors [50] or Intensity Fading (IF) Matrix Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) [51,52], provide reliable information about the presence of target interactors even in the highly heterogeneous context of natural extracts. Nevertheless, they have been rarely used for the identification of protease inhibitors in these sources.…”

Investigation of calmodulin-peptide interactions using matrix-assisted laser desorption/ionization mass spectrometry

“…Recent studies have suggested that the specific interactions between proteins and their peptide ligands could be deduced using IF-MALDI-MS, namely, introduction of a protein receptor could result in a selective decrease of the ion abundances of its peptide ligands compared with the nonbinding control [25][26][27][28][29][30][31][32][33][34].…”

“…Recently, Aviles and coworkers have developed an alternative strategy to detect the presence of noncovalent complexes in solution using intensityfading (IF-) MALDI-MS [25][26][27], which was based on a selective reduction of the ion abundance of the specific ligands after the addition of their receptor protein. The strategy was similar to the immunological procedures for identification of the epitopic peptides for antibodies introduced by the Downard group [28 -31] and has shown promising applications to detect several biological interactions [25][26][27][28][29][30][31][32][33][34][35][36][37]. Nevertheless, it should be noted that a nonbinding control is usually necessary to assess the specificity of binding assay in both direct MALDI-MS and IF-MALDI MS analysis [24 -37].…”