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purification
the total proteins from the examined as well as the control samples were extracted using the 2d-clean-up kit, and the purity was determined using bio-rad 2-d quant kit. about 2,000 µg of protein was loaded onto the ipg gel strip (ph 3-10) and then subjected to separation on a 12.5% sds-polyacrylamide gel. the efficiency of 2-d clean-up and subsequent proteomic analysis was assessed by the absence of staining, as well as by the presence of protein spots in gel strips from control and contaminated samples. after 2-d clean-up, the bradford method was used to determine the protein content of each sample [ ver. 7.0.0 software to identify the spots that were differentially expressed. the expression of the different proteins was compared to that in the control group for confirmation of possible contaminants. also the samples with a modified pattern and/or low concentration of proteins were discarded.
a total of 3,537 spots were detected from the gel images, of which 571 spots were significantly differentially expressed among the three groups of samples. these spots were excised from the gels and subjected to mass spectrometry (ms). however, some were too small to be analyzed, so the remainder were manually picked and then analyzed.
proteins
a large number of proteins were detected from the skin. the protein concentrations of the three sample groups were as follows: control (27.44 µg), ctx (23.12 µg), and mc (19.51 µg). the protein concentration of the blank/contaminated samples was 18.91 µg, which was 1/10th that of the control, while the total volume of the sample was the same. therefore, it can be concluded that the protein concentration of contaminated skin samples was significantly lower than that of the control samples. the protein spots from the differentially expressed proteins were identified by esi-ms/ms. upon comparing the protein spots among the control and contaminated samples, only three proteins were identified as possible contaminants in the control skin samples. similarly, one protein spot was identified from the ctx skin samples as a possible contaminant, and one protein spot was identified from the mc skin samples as a possible contaminant, but the ms analysis of the contaminated samples was not conclusive due to the presence of so many protein spots.
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