Characterization of peptides resulting from digestion of human skin elastin with elastase
Characterization of peptides resulting from digestion of human skin elastin with elastase
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DOI：
10.1002/prot.20643
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发表时间：
2005-11-15
影响因子：
2.9
通讯作者：
Neubert, RHH
中科院分区：
文献类型：
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作者：
Getie, M;Schmelzer, CEH;Neubert, RHH
几种病理性疾病与主要由弹性蛋白组成的弹性纤维异常有关。了解此类疾病的生化基础需要有关弹性蛋白一级结构的信息。由于弹性蛋白极不溶于水或任何有机溶剂，阻碍了其结构信息的获取，因此在本研究中，用弹性蛋白酶消化人类皮肤弹性蛋白以产生水溶性肽。串联质谱 (MS/MS) 实验分别使用传统电喷雾电离 (ESI) 和纳米 ESI 技术与离子阱和四极飞行时间 (qTOF) 质量分析仪结合进行。使用数据库搜索和/或从头测序从片段光谱中鉴定肽。首次确定了该酶的裂解位点以及人类皮肤弹性蛋白中脯氨酸羟基化的程度和位置。共鉴定出117个肽段，序列覆盖率为58.8%。据观察，测序区域中 25% 的脯氨酸残基被羟基化。弹性蛋白酶主要在氨基酸 Gly、Val、Leu、Ala 和 Ile 的 C 端裂解，在较小程度上裂解 Phe、Pro、Glu 和 Arg。我们的结果证实了之前的报告，即人类皮肤弹性蛋白缺乏外显子 26A 表达的氨基酸序列。蛋白质 2005；61：649-657。 (c) 2005 年 Wiley-Liss, Inc.
Several pathological disorders are associated with abnormalities in elastic fibers, which are mainly composed of elastin. Understanding the biochemical basis of such disorders requires information 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 elastin was digested with elastase to produce watersoluble peptides. Tandem mass spectrometry (MS/ MS) experiments were performed using conventional electrospray ionization (ESI) and nano-ESI techniques 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 sequencing. The cleavage sites of the enzyme and, for the first time, the extent and location of proline hydroxylation 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 hydroxylated. 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. (c) 2005 Wiley-Liss, Inc.