Increased keratin content detected by proteomic analysis of exhaled breath condensate from healthy persons who smoke  Elisabetta Gianazza, PhD, Luigi Allegra, MD, Enrica Bucchioni, MD, Ivano Eberini, PhD, Lina Puglisi, PhD, Francesco Blasi, MD, PhD, Claudio Terzano, MD, Robin Wait, PhD, Cesare R Sirtori, MD, PhD  The American Journal of Medicine  Volume 117, Issue 1, Pages 51-54 (July 2004) DOI: 10.1016/j.amjmed.2004.01.022

Figure Panel A (top): Two-dimensional electrophoretic (DE) fractionation of exhaled breath condensate (EBC) proteins—first dimension: nonlinear 4-10 immobilized pH gradient; second dimension: 4% to 20% polyacrylamide gradient. Cropped image: exhaled breath condensate proteins, pooled from 8 smokers, were freeze-dried and resolubilized in 8 M urea, and then applied at the cathode for isoelectric focusing. Proteins were fractionated by charge, and then by molecular size along orthogonal directions. Silver staining was performed avoiding cross-linking reagents to ensure compatibility with subsequent mass spectrometric analysis. Spots (marked 1 to 5) were excised from the gel, digested with trypsin, and analyzed by electrospray tandem mass spectrometry. Panel B (bottom): Silver-stained sodium dodecyl sulfate polyacrylamide (4% to 20% gradient) gels of exhaled breath condensate proteins. Consecutive samples: upper lane (1-11) from nonsmokers; middle and bottom lanes (12-38) from heavy smokers. Samples 37 and 38 were obtained from recently tracheostomized patients. Aliquots of 1 mL were freeze-dried, and the proteins carboxymethylated in the presence of 1% sodium dodecyl sulfate. The American Journal of Medicine 2004 117, 51-54DOI: (10.1016/j.amjmed.2004.01.022)