NOTE Characterization of new outer membrane proteins of Pseudomonas aeruginosa using a combinatorial peptide ligand library Mohamed Amine Ben Mlouka & Arbia Khemiri & Damien Seyer & Julie Hardouin & Philippe Chan Tchi Song & Emmanuelle Dé & Thierry Jouenne & Pascal Cosette Published online: 4 December 2014 # Springer-Verlag Berlin Heidelberg 2014 Abstract Most often, the use of ProteoMiner beads has been restricted to human serum proteins for the normalization of major proteins, such as albumin. However, there are other situations of interest in which the presence of major proteins would quench the signals of low abundance polypeptides. We propose the use of these beads for investigating the envelope of the gram-negative bacterium Pseudomonas aeruginosa. Initially, we performed comparative 2D electrophoresis to qualitatively evaluate the incidence of the normalization stage. This demonstrated a significant reduction of the major mem- brane proteins. Thereafter, using shotgun analysis, the same protein extract was targeted by using combinatorial peptide ligand library capture. This treatment yielded 154 additional outer membrane proteins (OMPs) uncovered by the study of the crude sample. Keywords Pseudomonas aeruginosa . Outer membrane proteins . Proteinnormalization . Combinatorialpeptideligand library Introduction Proteomics consists in the large scale study of proteins, focus- ing mainly but not restrictively on their expression level, post- translational modification, function, and localization. Today, it is recognized that genome sequencing alone is of limited value to obtain a complete understanding of gene function and role in cellular physiology. The proteome provides invaluable complementary information compared to genomics by char- acterizing the abundances and structures of gene products, but also post-translational modifications which are often key cel- lular processes, but that lay beyond our reach from genomic investigations. However, major challenges still prevent proteomics reaching exhaustivity in cells or biological fluids (e.g., hydro- phobicity, dynamic range). These difficulties favored the ap- pearance of fractionation approaches to narrow down the dynamic range and/or to reduce samples complexity. As an illustration, the use of solid-phase hexapeptide ligand libraries has demonstrated interesting potentialities [1]. Besides, though greatly improved in recent years [2], proteomics anal- ysis of membrane proteins remains problematic, because of their highly hydrophobic nature. This lack of solubility, ini- tially described in gel-based methods [3], remains or is even emphasized in methods using LC–MS [2]. In previous works [4, 5], we noticed on one- or two- dimensional electrophoresis (2-DE) gels of Pseudomonas Electronic supplementary material The online version of this article (doi:10.1007/s00216-014-8313-7) contains supplementary material, which is available to authorized users. M. A. Ben Mlouka : A. Khemiri : J. Hardouin : E. Dé : T. Jouenne : P. Cosette (*) CNRS UMR 6270, Laboratory Polymères, Biopolymères, Surfaces, University of Rouen, 76820 Mont-Saint-Aignan, France e-mail: pascal.cosette@univ-rouen.fr M. A. Ben Mlouka : A. Khemiri : J. Hardouin : E. Dé : T. Jouenne : P. Cosette Normandie Université, Esplanade de la paix, 14032 Caen Cedex 5, France M. A. Ben Mlouka : A. Khemiri : J. Hardouin : E. Dé : T. Jouenne : P. Cosette PISSARO proteomic facility, IRIB, University of Rouen, 76820 Mont-Saint-Aignan, France D. Seyer Laboratory ERRMECe, University of Cergy Pontoise - UFR Sciences et Techniques, 95302 Cergy Pontoise, France P. Chan Tchi Song INSERM, U982, 76820 Mont-Saint-Aignan, France Anal Bioanal Chem (2015) 407:1513–1518 DOI 10.1007/s00216-014-8313-7