Determination of Protein-Protein Interaction for Cancer Control via Mass Spectrometry and Nanoconductimetry of NAPPA SNAP Arrays: An Overview

Claudio Nicolini, Nicola Luigi Bragazzi and Eugenia Pechkova

Abstract

Background: Protein-protein interactions play a major role in Cancer Control and their detailed understanding by Label-Free Nanotechnology is essential especially within the framework of a personalized medicine-based approach.

Materials and Methods: We implemented an array of label-free nanobiotechnologies, including the Quartz Crystal Microbalance with Dissipation factor monitoring (QCM_D). We used it for the conductometric monitoring of an antiblastic (temozolomide) interacting with genes and proteins, such as MLH1, that represents a biomarker of the rate survival of patients suffering from brain tumors, outcome of chemotherapy and resistance to drug itself. We coupled the Nucleic Acid Programmable Protein Arrays (NAPPA) and the cell-free protein array with the quartz crystal microbalance technology. In another proof of principle, we coupled the NAPPA with the SNAP tag E. coli cell-free expression system. The goal is to analyze the protein-protein interaction using Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Bruker Ultraflex and “Protein synthesis Using Recombinant Elements” (PURE) system, thus avoiding the “black box” nature of the cell extract. The E. coli in vitro transcription/translation system (IVTT) in respect to the reticulocyte lysate (RRL) or human lysate (HL) is totally characterized and represents an advantage for the subsequent mass spectrometry (MS) analysis. An R Script for Mass Spectrometry Data Preprocessing before Data Mining (SpADS) provides the user with peak recognition and amplitude independent subtraction functions. The MS samples are obtained from SNAPNAPPA spots and printed on gold coated glass slides in higher density, in order to obtain an amount of protein appropriate for MS analysis.

Conclusion: We developed a coherent approach that overcome the drawbacks and pitfalls of the traditional laborious and time-consuming labeled and fluorescencebased experimental procedures. This, taken together with the unique properties of proteins obtained with Langmuir-Blodgett (LB)-based crystallography that can enable new strategies for drug design separately reported, defines our approach to cancer control.

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