Improved preparation of nasal lavage fluid (NLF) as a noninvasive sample for proteomic biomarker discovery ☆ Bodo Schoenebeck a , Caroline May b , Christian Güldner g , Gesine Respondek h , Brit Mollenhauer c , Guenter Hoeglinger d,e , Helmut E. Meyer i , Katrin Marcus f, ⁎ a Abteilung für Neuroanatomie und Molekulare Hirnforschung, Medizinische Fakultaet, Ruhr-Universitaet Bochum, 44801 Bochum, Germany b Abteilung für Medizinische Proteomik/Bioanalytik, Medizinisches Proteom-Center, Ruhr-Universitaet Bochum, 44801 Bochum, Germany c Paracelsus Elena-Klinik, 34128 Kassel, Germany d Lehrstuhl für Translationale Neurodegeneration, Technische Universität München, Germany e Deutsches Zentrum für Neurodegenerative Erkrankungen, 81377 München, Germany f Abteilung für Funktionelle Proteomik, Medizinisches Proteom-Center, Ruhr-Universitaet Bochum, 44801 Bochum, Germany g Department of Otolaryngology, Phillips Universität, Marburg, Germany h Department of Neurology, Technische Universität München, Munich, Germany and German Center for Neurodegenerative Diseases (DZNE), Munich, Germany i Leibniz-Institut für Analytische Wissenschaften -ISAS- e.V., Dortmund, Germany abstract article info Article history: Received 9 July 2014 Received in revised form 21 January 2015 Accepted 26 January 2015 Available online 10 February 2015 Keywords: Nasal lavage fluid 2D-gel electrophoresis Nasal lavage fluid (NLF) becomes more and more important as a noninvasive patient sample serving as a new opportunity to discover new biomarkers in diverse human diseases comprising mainly respiratory disorders. This was supported by the observation that the protein profile of NLF differs from conventional samples of i.e. whole blood, hence being capable to complement or even expand the so far biomarker index. Since sample acquisition and processing are the most crucial steps for a profound and sensitive identification we present here a modified protocol of NLF generation and measurement. We show that mild washing steps for sample generation followed by column-mediated concentration and acetone precipitation are appropriate steps to minimize serum leakage by concomitantly highlighting proteins which represent typical components of NLF. This is shown by separation of proteins via 2D-PAGE followed by LC-MS/MS as well as Gel-LC-MS/MS measurements of cut and digested protein spots/bands. Significance: For a better understanding of the molecular mechanisms underlying respiratory diseases NLF sam- ples are favored sources for protein research. NLF acquisition and sample processing were impaired so far by the problem of blood serum leakage and high salt content. Here, we present a modified protocol of NLF generation leading to the display of typical inventory of NLF proteins combined with reduced salt and serum contaminants. By this, both assay reproducibility and the detection of up- or down-regulated proteins reliably can be discovered in the case of biomarker screenings in a disease versus control design. This article is part of a Special Issue entitled: Neuroproteomics: Applications in Neuroscience and Neurology. © 2015 Elsevier B.V. All rights reserved. 1. Introduction In clinical research appropriate sample acquisition is the key step at the very beginning of almost all studies. Sample preparation must meet different needs in cases of sample volume, purity, reproducibility, sam- ple accessibility and informative value. Noninvasive patient samples are crucial for a profound and even fast monitoring of known reference values or biomarkers. In biomarker research, most studies have been conducted on whole blood or serum as examples for non-invasive sam- ples. Both have been investigated intensively [1–4]. As an alternative to theses sample sources, nasal lavage fluids (NLF) and bronchioalveolar lavage fluids (BALF) recently arose showing promising data for a sup- plementary way for biomarker discovery. Especially for NLF, multiple studies monitored alterations caused by disease states of the airways like asthma [5], sinusitis [6], cystic fibrosis [7], seasonal allergic rhinitis [8–10] or general respiratory disorders [11–14]. All these studies prove the validity of NLF as a means for the identification of biomarkers of diverse disease states. The investigation of NLF was recently adopted even for a mouse model of allergic rhinitis [15]. In general, differences in protein expression found in disease versus healthy control states emphasize the clinical relevance of NLF samples [16–19]. Biochimica et Biophysica Acta 1854 (2015) 741–745 Abbreviations: NLF, nasal lavage fluid ☆ This article is part of a Special Issue entitled: Neuroproteomics: Applications in Neuroscience and Neurology. ⁎ Corresponding author at: Functional Proteomics, Medizinisches Proteom-Center, Ruhr-Universitaet Bochum, MA/Room 4.59a, Universitaetsstrasse 150, D-44780 Bochum, Germany. Tel.: +49 234 32 28444; fax: +49 234 32 14554. E-mail address: Katrin.Marcus@rub.de (K. Marcus). http://dx.doi.org/10.1016/j.bbapap.2015.01.015 1570-9639/© 2015 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbapap