Characterization of Peptides Resulting from Digestion of Human Skin Elastin with Elastase M. Getie, C.E.H. Schmelzer, and R.H.H. Neubert * Institute of Pharmaceutics and Biopharmaceutics, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany ABSTRACT Several pathological disorders are associated with abnormalities in elastic fibers, which are mainly composed of elastin. Understanding the biochemical basis of such disorders requires infor- mation about the primary structure of elastin. Since the acquisition of structural information for elastin is hampered by its extreme insolubility in water or any organic solvent, in this study, human skin elas- tin was digested with elastase to produce water- soluble peptides. Tandem mass spectrometry (MS/ MS) experiments were performed using conventional electrospray ionization (ESI) and nano-ESI tech- niques coupled with ion trap and quadrupole time- of-flight (qTOF) mass analyzers, respectively. The peptides were identified from the fragment spectra using database searching and/or de novo sequenc- ing. The cleavage sites of the enzyme and, for the first time, the extent and location of proline hydroxy- lation in human skin elastin were determined. A total of 117 peptides were identified with sequence coverage of 58.8%. It has been observed that 25% of proline residues in the sequenced region are hy- droxylated. Elastase cleaves predominantly at the C-terminals of the amino acids Gly, Val, Leu, Ala, and Ile, and to a lesser extent at Phe, Pro, Glu, and Arg. Our results confirm a previous report that human skin elastin lacks amino acid sequences expressed by exon 26A. Proteins 2005;61:649 – 657. © 2005 Wiley-Liss, Inc. Key words: proline hydroxylation; peptide se- quencing; liquid chromatography (LC); tandem mass spectrometry (MS/MS); electrospray ionization (ESI); nano- electospray; cleavage sites; de novo sequencing INTRODUCTION Elastin is an important connective tissue protein that provides elasticity to organs, such as skin, lung, blood vessels, and ligaments. 1 It is among the most hydrophobic proteins known. Although there is some species variation, elastin from higher vertebrates, including human beings, contains over 30% Gly, and approximately 75% of the entire sequence is made up of just four hydrophobic amino acids (Gly, Val, Ala, Pro). Due to the extreme insolubility of elastin in water or organic solvents, research into the primary structure of elastin was hampered until the discovery of the water-soluble precursor, tropoelastin, which was first isolated from copper-deficient animals. The amino acid sequences of tropoelastins from various sources have been determined using molecular biological techniques to isolate and sequence elastin genes. Hu- man, 2,3 chicken, 4 bovine, 5,6 and rat 7 tropoelastin genes have all been sequenced and the amino acid sequences of the protein determined. Studies on the primary structure of elastin are very useful in helping to understand the biochemical basis of several pathological conditions, including solar elastosis, emphysema, aneurysm, and atherosclerosis, in which the mechanical and elastic properties of tissues are altered. However, due to its extreme insolubility, such studies are mainly focused on complete hydrolysis of the protein in a strongly acidic environment and analysis of the resulting cross-linked amino acids, desmosine and its isomer isodes- mosine, which are only found in elastin. 8 –10 This approach has been challenged by several factors, including the incomplete separation of the amino acids with the analyti- cal methods employed to date and the insufficient informa- tion that can be obtained about the entire elastin molecule only based on the cross-linked amino acids. Extraction of elastin from healthy and diseased tissue and comparison of the primary structures of the peptides resulting from enzymatic digestion of the protein could provide insight into the biochemical changes that occur due to the patho- logical conditions mentioned. Accomplishment of this task requires the identification of the resulting peptides by determining the primary sequences of these peptides. Automated Edman degradation has been widely used for the determination of the primary structure of peptides. However, it is now being increasingly replaced by mass spectrometric methods 11,12 such as electrospray ionization (ESI) 13 or nano-ESI 14 combined with quadrupole time-of- flight (qTOF) or ion trap mass spectrometry and by matrix-assisted laser desorption/ionization (MALDI)- TOF 15,16 by virtue of the following advantages: 1. The ability to perform sequence analysis of a peptide within a mixture because it can be mass-selected by the instrument; 2. Fragmentation of a molecular ion is possible even in the *Correspondence to: R.H.H. Neubert, Institute of Pharmaceutics and Biopharmaceutics, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck Str. 4, D-06120, Halle (Saale), Germany. Received 21 February 2005; Revised 27 April 2005; Accepted 28 April 2005 Published online 13 September 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/prot.20643 PROTEINS: Structure, Function, and Bioinformatics 61:649 – 657 (2005) © 2005 WILEY-LISS, INC.