纳升级反相液相色谱-串联质谱法分析蜈蚣提取蛋白质

Analysis of Extracted Proteins of Scolopendraby Nanoflow Reversed Phase Liquid Chromatography-Tandem Mass Spectrometry

结合聚丙烯酰胺凝胶电泳和纳升级反相液相色谱-串联质谱(nanoRPLC-MS/MS)法系统分析了蜈蚣提取蛋白质胶内酶解和直接酶解产物,并进行数据库检索鉴定蛋白质。建立的nanoLC分析条件为:上样8μL,经Chrom XP-C18毛细管柱(350μm×0.5 mm,3μm),2%乙腈(0.1%甲酸)-98%乙腈(0.1%甲酸)梯度洗脱90 min;采用ESI-Q-TOF MS,并在IDA采集模式下分析鉴定蜈蚣提取蛋白质。胶内酶解鉴定到72种蛋白质,直接酶解鉴定到97种蛋白质,二者含26种相同的蛋白质。通过数据库比对,分析了蜈蚣提取蛋白质的生物进程、细胞组分以及功能活性。得到蜈蚣蛋白质具有结合、酶催化、肌动等功能活性,其中结合活性的蛋白质占的比例较大,且多为能量代谢进程中的相关酶类。

Anovel nanoLC-MS/MS method coupled with SDS-PAGE was developed for analyzing extracted proteins of gel-based and gel-free digestion products from Scolopendra. NanoLC separation was performed on aChrom XP-C18 capillary column （350μm×0.5 mm,3μm）using a gradient elution program of 2% acetonitrile solution （containing 0. 1% formic acid） and 98% acetonitrile solution （containing 0.1% formic acid） as the mobile phase with a flow rate of 300 nL/min and theinjection volume of 8 μL. Quadruple-time-of- flight mass spectrometer （Q-TOF-MS） and electrospray ion source （ESI） were applied for qualitative analysis under the information dependent acquisition （IDA） mode. 72 kinds of proteins from gel-based method and 97 kinds of proteins from gel-free method were identified, both containing 26 kinds of common proteins. Based on the gene ontological analysis, the proteins identified forScolopendra were differentially displayed according tocellular component, biological process and molecular functions. The function of proteins included binding activity, enzyme catalytic activity, motor activity andantioxidant activity, in which binding activity was the major molecular functions involved in the energy metabolism.