A new methodology for monitoring the activity of cdMMP-12 anchored and freeze-dried on Au (111)

Abstract

Matrix metalloproteinases (MMPs) are cell-secreted soluble and membrane-tethered enzymes that are capable of degrading extracellular matrix proteins, but also can process a number of bioactive molecules. They are involved in the cleavage of cell surface receptors, but are also thought to play a major role on cell behaviors as well as in diverse physiological and pathological processes, including embryonic development, wound repair, inflammatory diseases, and cancer. For these reasons, it is obvious that a control over MMPs activity is highly desirable. Consequently, the frantic search for new inhibitors has been coupled to the development of high-throughput methods able to rapidly screen the effect of possible MMP inhibitors on the activity of these enzymes. In this scenario, solid-state-based methods play a major role because of their compatibility with array formats that are able to extract more information from smaller sample volumes and offer some important advantages that are not available in the standard solution assays. In this work, the catalytic domain of MMP-12 was immobilized on a gold substrate and the surface coverage was measured by FT-SPR experiments. A new experimental procedure was developed to freeze-dry the anchored molecules and their activity was measured by ESI-MS. The kinetics parameters obtained for the immobilized enzyme are in good accordance with those reported for similar systems in solution. Inhibition of the immobilized molecules was also carried out, demonstrating the applicability of the method for rapid screening of MMP inhibitors. (J Am Soc Mass Spectrom 2007, 18, 961-969)

New glycoside derivatives of carnosine and analogs resistant to carnosinase hydrolysis: Synthesis and characterization of their copper(II) complexes

“…Sample water solutions were injected into the ion source without the addition of any other solvent at a flow rate of 5 μL/ min, using nitrogen as the drying gas. All the other experimental parameters were the same as described elsewhere [43,44]. Xcalibur software was used for the elaboration of mass spectra.…”

Retinoid signaling competence and RARβ‐mediated gene regulation in the developing mammalian telencephalon

“…Solid templates also represent a very powerful approach for protein studies that are not available in the standard solution assays, such as the possibility to recycle the studied molecules, a tag‐free working environment, high throughput, and the improved discovery of protein‐binding partners through protein‐affinity interactions (Grasso et al, 2006). In vitro , the use of MS is particularly useful when the substrate cleavage sites of different Aβ‐degrading enzymes must be identified (Yan et al, 2006; Grasso, Rizzarelli, & Spoto, 2007), or the activities of the latter must be monitored (Crouch et al, 2009; Grasso et al, 2009a), and solid‐state assays have proven to be particularly useful for such purposes (Grasso et al, 2005, 2007; Toropygin et al, 2008). In the case of solid‐state assays, a key advantage of MALDI over ESI is the static nature of the ionization technique, a property that makes MALDI easily amenable to signal detection from microarray substrates (as opposed to the liquid probe introduction for ESI).…”

A neglected modulator of insulin-degrading enzyme activity and conformation: The pH

“…IDE was immobilized on a COOH5 biosensor chip from ICx Nomadics (Oklahoma City, USA). Briefly, covalent immobilization was obtained by amine coupling of the lysine-free amino groups and terminal amines of the protein, as described elsewhere [36][37][38]. Particularly, 400 μL of IDE solution at 100 μg/mL in 10 mM acetate buffer, pH 3.8, was used for the immobilization on a previously activated surface having reactive succinimide ester groups obtained by using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) solution, freshly prepared ((EDC) = 0.4 M, (NHS) = 0.1 M).…”