Method Research On Quantitative Analysis Of Proteins In Human Serum Based On Inductively Coupled Plasma Mass Sepctrometry Hyphenated Techniques

Transferrin(Tf) and albumin(Alb), as two important human serum proteins, play key roles in diagnosis disease, such as inflammation, coronary heart disease, anemia, discovery of disease markers, and mechanistic research on pathological conditions. For accurate quantification of human serum transferrin and albumin, method research based on inductively coupled plasma mass spectrometry (ICP-MS) hyphenated techniques is of great significance, helps to promote the measurement level of life science in our country, and gains momentum for measurement traceability study.The main contents in this thesis included,(1) Establishment of quantitative method using HPLC-ID-ICP-MS:Four mixed standard proteins, serving as model proteins, were used to develop the method based on post-column isotope dilution in combination with high performance liquid chromatography coupled to ICP-MS.(2) Optimization of HPLC condition in human serum:Separation conditions such as chromatographic column, time program, mobile phase were adjusted to achieve completely separation of Tf and Alb in human serum.(3) Quantitative analysis of Tf and Alb:We researched the application of established HPLC-ID-ICP-MS method in quantification of Tf and Alb. Tf was absolutely quantified via both sulfur and iron by introducing isotopically enriched34S and54Fe spikes together, and the concentrations of Tf and Alb via sulfur were simultaneously determined by altering the flow rate of enriched34S isotope solution. The proposed HPLC-ID-ICP-MS methods were tested for the analysis of human serum protein certified reference material (ERM-DA470k/IFCC).(4) Optimization of polyacrylamide gel electrophoresis (PAGE) separation condition in human serum.(5) Establishment of isotope dilution after electrophoresis:Three different addition ways of enriched34S isotope solution were determined by laser ablation ICP-MS and the results showed that protein strip soaking with enriched34S isotope solution after gel electrophoresis realized co-existence of34S spike and natural sulfur in the gel. Different concentrations of34S spike and soaking times were discussed to obtain optimum isotope dilution condition.(6) Quantification of Tf and Alb in human serum was carried out by post-electrophoresis isotope dilution in combination with PAGE-LA-ICP-MS and the method was validated with ERM-DA470/IFCC.In this work, we established quantitative methods of HPLC-ID-ICP-MS, PAGE-LA-ID-ICP-MS based on ICP-MS hyphenated techniques and optimized separation conditions of human serum by HPLC and PAGE. Then, we used the two methods to quantify Tf and Alb in human serum and validated the methods with human serum protein certified reference material (ERM-DA470k/IFCC). All the results showed that the established methods were both suitable for accurate quantification of proteins in samples with complex matrix such as human serum.