Comparison of methods for enriching urine proteins / 生物工程学报

The abundance of proteins in human urine is low and easily to be masked by high-abundance proteins during mass spectrometry analysis. Development of efficient and highly selective enrichment methods is therefore a prerequisite for achieving deep coverage of urine protein markers. Notably, different experimental methods would affect the urine protein enrichment efficacy and the coverage of urine proteome. In this study, ultrafiltration, nitrocellulose membrane enrichment and saturated ammonium sulfate precipitation were used to process 10 mL urine samples from five healthy volunteers and five bladder cancer patients. The urine proteins were enriched and separate by SDS-PAGE to compare the purification efficiency of different methods. Moreover, the peptide identification effects of different purification methods were analyzed by mass spectrometry to determine the best method for enriching urine protein histones. Saturated ammonium sulfate precipitation method outperformed the ultrafiltration and the nitrocellulose membrane enrichment methods in terms of the protein enrichment efficacy and quality. The interference of highly abundant albumin was reduced, whereas the amount of low-abundance protein was increased, and the sensitivity of mass spectrometry identification was increased. The saturated ammonium sulfate precipitation method may be applied for large-scale urine processing for screening clinical diagnostic markers through proteomics.

Preparation of human transferrin and verification of the antigenic activity / 国际检验医学杂志

Objective Non-affinity methods were used to purify transferrin(TRF)with high purity from human serum,and the immunogenicity of TRF was evaluated by immunizing New Zealand rabbits.Methods Firstly,TRF was extracted from serum by precipitation with ammonium sulfate and then purified by two-step anion exchange chromatography.Results SDS-PAGE purity of the prepared TRF was similar with the control pure product,and the HPLC purity reached to 96.8% with a yield of 78.6%.For the same batch of TRF sam-ple,the ratio between the activity concentration determinated using TRF kit(immunoturbidimetry method) and the protein concentration determinated using uv-spectrophotometric method was about 0.95,which indica-ted that the prepared TRF for antigen could react well with the TRF antibody included in the TRF kit.Final-ly,New Zealand rabbits were immunized using the purified TRF,and the titer of the rabbit anti-serum could reach 1:128 000 after four time immunization,which also indicated that the prepared TRF had good immuno-genicity.Conclusion The TRF with high purity had good antigen reactivity and immunogenicity to prepare anti-T RF antibody by immunizing rabbits,w hich could provide qualified materials for the production of T RF kit(immunoturbidimetry method).