Nousiainen M, Rafn K, Skou L, Roepstorff P, Andersen S O
Department of Molecular Biology, Odense University, Denmark.
Comp Biochem Physiol B Biochem Mol Biol. 1998 Jan;119(1):189-99. doi: 10.1016/s0305-0491(97)00306-4.
Proteins from the calcified exoskeleton of the lobster, Homarus americanus, were extracted and separated by two-dimensional gel-electrophoresis. Electroblotting the proteins onto polyvinylidene difluoride (PVDF) membranes followed by sequence determination gave 16 N-terminal amino-acid sequences and revealed that further eight proteins were N-terminally blocked. The relative molecular mass, M(r), was obtained for most of the electrophoretically separated proteins by means of matrix-assisted laser desorption mass spectrometry (MALDIMS) after electroelution from Coomassie-stained two-dimensional polyacrylamide gels. Eleven proteins were purified from extracts of the exoskeleton by low pressure ion exchange chromatography and reversed-phase high performance chromatography, and their sequences were determined by combined use of Edman degradation and mass spectrometry. Good agreement was obtained between the M(r)-values measured by mass spectrometry and those calculated from the sequences. Five of the sequenced proteins contain two copies of a previously observed 18-residue sequence motif, while a couple of the remaining sequences show similarity to sequences of exoskeletal proteins from shrimps and spiders. Only limited similarity to insect cuticular proteins was observed.
从美洲螯龙虾（Homarus americanus）钙化外骨骼中提取蛋白质，并通过二维凝胶电泳进行分离。将蛋白质电印迹到聚偏二氟乙烯（PVDF）膜上，随后进行序列测定，得到了16个N端氨基酸序列，并发现另外8种蛋白质的N端被封闭。通过基质辅助激光解吸质谱（MALDIMS）从考马斯亮蓝染色的二维聚丙烯酰胺凝胶上电洗脱后，获得了大多数经电泳分离蛋白质的相对分子质量（M(r)）。通过低压离子交换色谱和反相高效液相色谱从外骨骼提取物中纯化了11种蛋白质，并结合埃德曼降解和质谱法测定了它们的序列。质谱法测得的M(r)值与根据序列计算得到的值之间取得了良好的一致性。5种已测序的蛋白质含有两个先前观察到的18个残基序列基序的拷贝，而其余一些序列与虾和蜘蛛外骨骼蛋白质的序列具有相似性。仅观察到与昆虫表皮蛋白的有限相似性。
