1. Introduction The conventional mass spectrometry (MS) strategy for protein analysis, the so-called bottom-up approach, is based on enzymatic digestion, which renders a collection of peptides to be identifed by electrospray (ESI) MS through a peptide mass fngerprint [1]. A more comprehensive alternative also includes the information acquired by collision-activation dissociation (CAD) techniques for peptide sequencing in tandem MS (MS/ MS) or multistage MS (MS n ) [2]. Although the method has been used very successfully in many instances [1-4], it exhibits a series of disadvantages, among which the many laborious and time-consuming steps; diffculties Central European Journal of Chemistry * E-mail: alina.zamfr@uav.ro # These authors have contributed equally 1 Department of Chemical and Biological Sciences, “Aurel Vlaicu” University of Arad, 310130 Arad, Romania 2 Mass Spectrometry Laboratory, National Institute for Research and Development in Electrochemistry and Condensed Matter, 300224 Timisoara, Romania 3 Physics Department, West University of Timisoara, 300223 Timisoara, Romania 4 Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, Department of Chemistry, Zukunftskolleg, University of Konstanz, 78457 Konstanz, Germany 5 Chemistry Institute of Romanian Academy, 300223 Timisoara, Romania Corina Flangea 1# , Catalin Schiopu 1# , Florina Capitan 1# , Cristina Mosoarca 2,3 , Marilena Manea 4 , Eugen Sisu 5 , Alina D. Zamfr 1* Fully automated chip-based nanoelectrospray combined with electron transfer dissociation for high throughput top-down proteomics Research Article Abstract: © Versita Sp. z o.o. Received 16 May 2012; Accepted 16 August 2012 Keywords: Chip-electrospray mass spectrometry • Electron transfer dissociation • Peptides • Proteins • Top-down analysis The conventional protocol for protein identifcation by electrospray ionization mass spectrometry (MS) is based on enzymatic digestion which renders peptides to be analyzed by liquid chromatography-MS and collision-induced dissociation (CID) multistage MS, in the so-called bottom-up approach. Though this method has brought a signifcant progress to the feld, many limitations, among which, the low throughput and impossibility to characterize in detail posttranslational modifcations in terms of site(s) and structure, were reported. Therefore, the research is presently focused on the development of procedures for effcient top-down fragmentation of intact protein ions. In this context, we developed here an approach combining fully automated chip-based-nanoelectrospray ionisation (nanoESI), performed on a NanoMate robot, with electron transfer dissociation (ETD) for peptide and top-down protein sequencing and identifcation. This advanced analytical platform, integrating robotics, microfuidics technology, ETD and alternate ETD/CID, was tested and found ideally suitable for structural investigation of peptides and modifed/functionalized peptides as well as for top-down analysis of medium size proteins by tandem MS experiments of signifcantly increased throughput and sensitivity. The obtained results indicate that NanoMate-ETD and ETD/CID may represent a viable alternative to the current MS strategies, with potential to develop into a method of routine use for high throughput top-down proteomics. Cent. Eur. J. Chem. • 11(1) • 2013 • 25-34 DOI: 10.2478/s11532-012-0130-2 25