| Identification and comparative quantification between protein samples were performed using isobaric tags for relative and absolute quantification(iTRAQ), which could move forward to compare protein expression levels among more complex samples. Protein samples subjected to enzymatic digestion of trypsin were labeled with one of several chemically identical iTRAQ tags respectively before pooled, and then the mixed peptides labeled were separated and analyzed via strong cation exchange (SCX) chromatography followed by nano reverse phase (RP) chromatography combined with mass spectrometry (MS). A total of 54 unique peptides and 6 standard proteins were identified successfully with 95% confidence, including Escherichia coli 茁-galactosidase, 茁-lactoglobulin, chick lysozyme. Human serotransferrin in sample A did not show significant difference at protein quantitative expression levels compared with that in sample B, whereas the quantity of bovine serum albumin (BSA) and 琢-lactalbumin in sample B cut down to half of that in sample A.The results correspond to the designed experiments, and the repeatability test shows that the approach is stable and reproducible. It reveals the technique is well suitable for the identification and comparative quantitative studies of protein expression levels, in addition, which can significantly increase the investigative throughput of protein mixtures. |
