Mass Spectrometry and Florescence Analysis of Snap-Nappa Arrays Expressed Using E. coli Cell_Free Expression System

Abstract

We present the analysis of an innovative kind of self-assembling protein microarray, the "Nucleic Acid Programmable Protein Array" (NAPPA), express with the SNAP tag in E.coli coupled self free expression system. The goal is to develop a standardize procedure to analyze the protein protein interaction occurred on NAPPA array combining label free Mass Spectrometry (MS) and fluorescence technology for protein microarray. We employ in the process "Protein synthesis Using Recombinant Elements" (PURE) system. For the first time an improved version of NAPPA, that allows for functional proteins to be synthesized in situ -with a SNAP tag -directly from printed cDNAs just in time for assay, has been expressed with a novel cell-free transcription/translation system reconstituted from the purified components necessary for Escherichia coli translation -the PURE system -and analyzed both in fluorescence and in a label free manner by four different mass spectrometers, namely three Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF), a Voyager, a Bru ker Autoflex and a Bruker Ultraflex, and Liquid Chromatography-Electrospray Ionization Mass Spectrometry (LC-ESI-MS/MS). Due to the high complexity of the system, an ad hoc bioinformatic tool has been needed to develop for their successful analysis. The contemporary fluorescence analysis of NAPPA, expressed by means of PURE system, has been performed to confirm the improved characterization of this new NAPPA-SNAP system.

Drug-Protein Interactions for Clinical Research by Nucleic Acid Programmable Protein Arrays-Quartz Crystal Microbalance with Dissipation Factor Monitoring Nanoconductometric Assay

“…In this study, we introduced a new application of our previously described NAPPA-based nanoconductometric sensor, (Nicolini et al, 2012a;2012b;Nicolini et al, 2013), which combined with Mass Spectrometry using SNAP arrays (Nicolini et al, 2013) has been extended to cancer studies (Bragazzi et al, 2014a), and in this context has been used to clinically screen patients respondent to TMZ from those refractory to this drug. We performed a positive control (TMZ plus MLH1 protein), a negative control (TMZ plus BRIP1 protein) and a multi-gene experiment (TMZ plus BRIP1&MLH1 being co-expressed), showing that we are able to properly perform pharmacoproteomics tasks, discriminating each protein and drug unique conductance curve as well as their interactions, even in the presence of multi-proteins being immobilized.…”

“…In the last years, the evolution of the nanobiotechnologies applied to proteins, namely proteomics, both structural and functional and specifically the development of more sophisticated protein arrays, has enabled scientists to investigate protein interactions and functions with an unforeseeable precision and wealth of details (Nicolini et al, 2012a;2012b;Nicolini et al, 2013). Moreover, protein arrays can be coupled with label-free approaches: The so-called cell-free protein arrays (Bragazzi et al, 2014b;Dixon, 2008;Fee, 2013;Hunter, 2009;Spera et al, 2013).…”

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

“…Also, the ongoing research in the field of MS coupled with NAPPA and SNAP is giving, as shown recently [26,30], encouraging results in the study of protein-protein interaction, a topic relevant for cancer control. We strongly believe that these efforts can be precious in the field of personalized oncology.…”

“…Another innovative approach we developed is given by coupling NAPPA with the SNAP tag E. coli coupled cell-free expression system and Mass Spectrometry (MS) (in particular, the Matrix Assisted Laser Desorption Ionization Time-ofFlight (MALDI-TOF) Bruker Ultraflex) with the goal of implementing a standardized procedure of identification of biomarkers in clinical settings and to analyze the proteinprotein interaction occurred on NAPPA array using label-free technology [26]. We employed the process "Protein synthesis Using Recombinant Elements" (PURE) system, which, due to its high complexity, needs ad hoc in-house developed bioinformatics tools to be analyzed.…”

Microarray-based Functional Nanoproteomics for an Industrial Approach to Cancer. II Mass Spectrometry and Nanoconductimetry

“…On the light of the sixteen recently published experimental papers, referred in the last section [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16], we have successfully completed the feasibility study on Microarray-based Functional Nanoproteomics for an Industrial Approach to Cancer.…”