Abstract: 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.

Key words: RhD blood group protein sequence, Erythrocyte protein, Immunoprecipitation method, Mass spectrometry