Mass spectrometry and florescence analysis of Snap-Nappa arrays expressed using E. coli cell_free expression system

Claudio Nicolini, Rosanna Spera, Fernanda Festa, Luca Belmonte, Shaorong Chong, Eugenia Pechkova, Joshua Labaer

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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.

Original languageEnglish (US)
Article number1000181
JournalJournal of Nanomedicine and Nanotechnology
Volume4
Issue number5
DOIs
StatePublished - Dec 1 2013

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All Science Journal Classification (ASJC) codes

  • Bioengineering
  • Medicine (miscellaneous)
  • Biomedical Engineering
  • Pharmaceutical Science

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