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

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.complement to labeling methods [7,8].Among all these techniques, that usually cannot reveal the identity of interaction proteins, MS is powerful to provide chemical and structural information that is difficult to obtain through other means.Combining MS and other surface techniques could offer new dimensions in protein analysis [5,8].The integration of microarrays with MS has generated a powerful new tool to deal with the problems in protein analysis and identification area.The most successful example is the ProteinChip ® System of Ciphergen Biosystems Inc.The design of the ProteinChip ® array was originally derived from chromatography and is divided into two groups according to its surface characteristics; the array reader is a SELDI-TOF-MS instrument equipped with a pulsed UV nitrogen laser source [8].Also Nedelkov et al. [9] coupled BIACORE with MS to demonstrate its feasibility in detecting multiple protein-protein interactions.In our research we employed two different mass spectrometry (MS) techniques, the Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) MS and Liquid Chromatography-Electrospray Ionization MS (LC-ESI-MS) (Figure 1).In a previous research we carried out a feasibility study of MALDI-