Comparison of two tandem mass spectrometry-based methods foranalyzing the proteome of healthy human lens fibers

[摘要] Purpose: To establish a method for studying the proteome of humanlens fibers and to provide the proteome database of lens fibers fromhealthy male adults.Methods: We compared two liquid chromatography tandem massspectrometry (LC-MS/MS)-based methods for studying the proteome ofhealthy adult human lens fibers. Total proteins were extracted frompooled lens fibers of 12 healthy male adult donors. In one method, thetotal proteins were digested with trypsin, and the derived peptides wereanalyzed by strong cation exchange (SCX) coupled with reverse phaseliquid chromatography tandem mass spectrometry (RPLC-MS/MS). In theother method, proteins were first resolved by sodium dodecyl sulfatePAGE (SDS-PAGE) and then in-gel digested with trypsin, and the peptideswere analyzed by RPLC/MS/MS. The tandem mass spectra of positive resultswere quality controlled by advanced mass spectrum scanner (AMASS)software. The peptide false positive rate was estimated using thereverse database searching method.Results: A total of 68 proteins from lens fibers were identifiedusing these two methods based on at least two different peptide matcheswith reliability of over 97% for each peptide. Among these proteins, 43were detected by both methods, one was detected only by SCX-RPLC/MS/MS,and 24 were detected only by SDS-PAGE-RPLC-MS/MS.Conclusions: The data clearly indicated that the SDS-PAGE-RPLC-MS/MSmethod was more suitable than the SCX-RPLC-MS/MS method for analyzinglens fiber proteome. This work greatly expanded the proteome database ofhuman lens fibers, and the results provided a reference for futurestudies to detect aging-related and cataract-related changes in humanlens fibers proteins.

[学科分类] 生物化学/生物物理