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Comparison of two tandem mass spectrometry-based methods for analyzing the proteome of healthy human lens fibers.

PURPOSE
To establish a method for studying the proteome of human lens fibers and to provide the proteome database of lens fibers from healthy male adults.


METHODS
We compared two liquid chromatography tandem mass spectrometry (LC-MS/MS)-based methods for studying the proteome of healthy adult human lens fibers. Total proteins were extracted from pooled lens fibers of 12 healthy male adult donors. In one method, the total proteins were digested with trypsin, and the derived peptides were analyzed by strong cation exchange (SCX) coupled with reverse phase liquid chromatography tandem mass spectrometry (RPLC-MS/MS). In the other method, proteins were first resolved by sodium dodecyl sulfate PAGE (SDS-PAGE) and then in-gel digested with trypsin, and the peptides were analyzed by RPLC/MS/MS. The tandem mass spectra of positive results were quality controlled by advanced mass spectrum scanner (AMASS) software. The peptide false positive rate was estimated using the reverse database searching method.


RESULTS
A total of 68 proteins from lens fibers were identified using these two methods based on at least two different peptide matches with reliability of over 97% for each peptide. Among these proteins, 43 were 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/MS method was more suitable than the SCX-RPLC-MS/MS method for analyzing lens fiber proteome. This work greatly expanded the proteome database of human lens fibers, and the results provided a reference for future studies to detect aging-related and cataract-related changes in human lens fibers proteins.