Bijar Ghafouri Bengt Ståhlbom ChristerTagesson Mats Lindahl Division of Occupational and Environmental Medicine, Linköping University, Linköping, Sweden Newly identified proteins in human nasal lavage fluid from non-smokers and smokers using two-dimensional gel electrophoresis and peptide mass fingerprinting Human nasal lavage fluids (NLFs) were analyzed with two-dimensional gel electro- phoresis (2-DE) and proteins were identified with peptide mass fingerprinting using matrix-assisted laser desoption/ionization time of flight mass spectrometry. In some cases, the identification was verified by analysis of post-source decay fragmentation spectra. Many of the identified proteins were new forms or fragments of previously found proteins (e.g. albumin, lactoferrin, cystatin, calgranulin, von Ebners gland protein and palate lung nasal epithelium clone), while others were proteins that have previously been indicated by 2-DE image matching or immunoblots (e.g. apolipoprotein AI, lyso- zyme C, and Clara cell secretory protein). Some new proteins, not shown before in 2-DE patterns of NLF were also found, e.g. mammaglobin B, b 2 -microglobulin and immunoglobulin J chain. Of the identified NLF proteins many appear to be involved in inflammatory and immune responses. A study was therefore conducted to investigate if the levels of these proteins were changed in smokers compared to nonsmokers. It was found that NLF from smokers contained decreased levels of Clara cell secretory protein, and increased proportions of a truncated variant of lipocortin-1, three acidic forms of a 1 -antitrypsin, and one phosphorylated form of cystatin S. Furthermore, NLF from smokers contained increased proportions of a new variant of palate lung nasal epithelium clone (PLUNC), a recently identified airway irritation marker. The results demonstrate that 2-DE of NLF may be used to assess alterations of proteins or post- translationally modified proteins in smokers. Clara cell secretory protein (CC 16, CC 10) and lipocortin-1 are two anti-inflammatory, phospholipase A 2 inhibitory proteins, and a 1 -antitrypsin and cystatin S are two proteinase inhibitors. Changed levels of these proteins may therefore be of importance to the airway inflammation caused by smok- ing. The results also support the notion that PLUNC is involved in inflammatory responses in the upper airways. Keywords: Nasal lavage fluid / Smokers / Two-dimensional gel electrophoresis / Mass spectro- metry PRO 0151 1 Introduction Nasal lavage fluid (NLF) contains a large number of proteins that altogether comprise a potential source for detecting and characterizing biochemical alterations associated with airway diseases [1]. Being a first line of defence, the nasal mucosa is constantly under the influence of different environmental agents such as irritating particles and chemi- cals, microorganisms and allergens, that may trigger inflam- matory and immune responses of the airways. For example, tobacco smoke is an established risk factor for rhinitis and asthma [2]. By using nasal lavage fluid samples from the upper airways, it may be possible to monitor protein changes that not only reflect the upper airways but also give information of processes in the lower airways [3, 4]. We have previously demonstrated that 2-DE patterns of human NLF may be used to detect differences in protein expression in subjects with asthma and rhinitis and in workers exposed to irritating chemicals [5–7]. So far, we have identified the proteins mainly by pattern matching with human plasma [1], immunoblotting [6] and by N-terminal amino acid sequencing [7]. However, mass spectrometry to determine peptide profiles after trypsin cleavage (peptide mass fingerprinting) is now the method of choice in order to quickly identify proteins, and map- ping of proteins in this way after 2-DE has become a cen- tral tool in linking the proteome to the human genome [8]. Correspondence: Dr. Mats Lindahl, Occupational and Environ- mental Medicine, Faculty of Health Sciences, Linköping Univer- sity, S-581 85 Linköping, Sweden E-mail: mats.lindahl@ihm.liu.se Fax: +46-13-145831 Abbreviations:CC16, Clara cell secretory protein 16; NLF , nasal lavage fluid; PLUNC, palate lung nasal epithelium clone 112 Proteomics 2002, 2, 112–120 ª WILEY-VCH Verlag GmbH, 69451 Weinheim, 2002 1615-9853/02/0101–112 $17.50+.50/0