The current explosion of emerging technologies of proteomics is expected to discover biomarkers and mechanisms of diseases. To define the difference in protein profile among experimental samples or in diseased tissues, the procedures in proteomic analysis should be considered to avoid loss of proteins with low abundance and to minimize experimental errors and contamination of keratins. In the present study, we examined and improved several procedures to reduce the protein loss during sample handling and storage. Microtubes from several companies were used to aliquot and store samples for MS analysis. We found several microtubes brought about high-level noise or high absorption of peptides. To minimize human keratin contamination we aimed to determine the process or procedure in which the keratin contamination was easy to happen during various steps of sample preparation from 2D gel electrophoresis to in gel digestion steps. We found that charge of static electricity on microtubes used for sample preparation was essential for adhesion of human keratin from air dust. Therefore, we modified the procedures to shorten the time for sample preparation by using dispensers for liquid handling and a vacuum set for discarding liquid. To keep our fingertips clean during every procedure, fingers were washed in distilled water or Milli-Q water frequently, and the vacuum set was applied to remove air dust adhered to the microtubes. By comparing the conventional procedures with our modified ones for the keratin contamination, the most critical step for avoiding keratin contamination was in-gel digestion step. By using our modified procedures, we could identify very low abundant proteins and avoid the human keratin contamination, indicating that our procedure is a practically good and easy way to minimize keratin contamination and to identify small copies proteins by mass spectrometry.
