免疫沉淀法与质谱联用检测RHD蛋白序列方法学的建立

RHD Protein Sequences by Immunoprecipitation and Mass Spectrometry

目的建立人类红细胞RHD蛋白序列分析的方法,为检测RhD血型基因在红细胞膜上抗原量的表达奠定基础。方法随机选取10例无偿献血者的抗凝血5 mL,均为已知RhD阳性,对标本中的Rh血型抗原蛋白进行免疫沉淀。采用蛋白酶解法从红细胞膜中提取RH蛋白并定量。经蛋白酶切割后,利用LC-MS质谱平台对Rh蛋白序列进行鉴定。结果建立了检测RHD蛋白序列的方法,制定出适合检测RHD蛋白的标准曲线,得到10例样本的RHD蛋白浓度,鉴定到的RHD目标蛋白序列与Blast平台检索到的RHD蛋白片段序列吻合度达100%,获得红细胞膜RHD蛋白肽段二级图谱。结论免疫沉淀法与质谱联用可以鉴定红细胞RHD蛋白的氨基酸序列,为检测RhD抗原在红细胞上的表达量奠定基础,为精确鉴定RhD血型提供可行的方法,具有广泛的应用价值。

Objective We established a method of sequence analysis for human erythrocyte Rhesus D(RhD)blood group protein to lay a foundation for identifying the expression of RhD blood group gene in erythrocyte membrane.Methods Five mL of anticoagulant blood from 10 blood donors with RhD positive were randomly selected.The Rh blood group antigen protein was immunoprecipitated.Rh(Rhesus)protein was extracted from erythrocyte membrane by protease hydrolysis and quantified.After being cleaved by protease,the Rh protein sequences were identified by mass spectrometry(LC-MS).Results The method for detecting RhD protein sequence was established and the standard curve of RhD protein was developed.The concentrations of RhD proteins in 10 samples were obtained.The RhD target protein and RhD protein fragment from Basic Local Alignment Search Tool(BLAST)platform exhibited 100%sequence identity,and a secondary mapping of RhD protein peptide in the erythrocytic membrane was obtained.Conclusions Immunoprecipitation coupled to mass spectrometry can define amino acid sequence of RhD protein in red blood cells to lay a solid foundation for verifying RhD antigens expressed on red blood cells.It can accurately determine RhD blood group and has a very wide application.