【摘要】 随着家蚕基因组图谱的发布，家蚕的研究逐渐进入后基因组时代，家蚕蛋白质组样品前处理方法没有统一的标准，鉴定到的蛋白质数量差异较大.基于超高分辨率质谱仪，建立冰上组织研磨器研磨提取蛋白质的方法，能极大地避免材料的损失，具有稳定和快速的优点.通过对不同提取试剂的摸索，发现尿素+DTT对家蚕组织样品的提取效果最好.通过对上样量和上样时间的摸索，确定最佳上样量为10μL,最佳上样时间为120 min.利用90%序列相似性原则构建非冗余家蚕数据库Strealine,在家蚕5龄第3 d脂肪体材料中，使用该库能鉴定到1 910种蛋白质.进一步采用反相色谱柱对肽段进行分级，将鉴定到的蛋白数量提升至3 986种.更多还原

【Abstract】 With the release of the silkworm genome map, research on silkworm has gradually entered the post-genomic era. There is no uniform standard for sample-pretreatment methods for silkworm proteome samples, and the number of proteins can be identified varies greatly. This study presents a novel method for protein extraction using a tissue grinder on ice. The proposed extraction method effectively minimizes material loss and offers improved stability and speed compared to traditional methods. After exploring different extraction reagents, it was found that urea with DTT has the best extraction effect on silkworm tissue samples. By exploring loading volumes and loading times, it was determined that the optimal loading volume was 10 μL and the optimal loading time was 120 min. Using the principle of 90% sequence similarity, a non-redundant silkworm database Strealine was constructed. Using this database, 1 910 proteins can be identified in the fat body material of silkworm on the third day of fifth instar. Reversed-phase chromatography columns were further used for peptide fractionation, the number of identified proteins increased to 3 986.更多还原