摘要
In this study we systematically analyzed the elution condition of tryptic peptides and the characteristics of identi?ed peptides in reverse phase liquid chromatogra- phy and electrospray tandem mass spectrometry (RPLC-MS/MS) analysis. Fol- lowing protein digestion with trypsin, the peptide mixture was analyzed by on-line RPLC-MS/MS. Bovine serum albumin (BSA) was used to optimize acetonitrile (ACN) elution gradient for tryptic peptides, and Cytochrome C was used to retest the gradient and the sensitivity of LC-MS/MS. The characteristics of identi?ed peptides were also analyzed. In our experiments, the suitable ACN gradient is 5% to 30% for tryptic peptide elution and the sensitivity of LC-MS/MS is 50 fmol. Analysis of the tryptic peptides demonstrated that longer (more than 10 amino acids) and multi-charge state (+2, +3) peptides are likely to be identi?ed, and the hydropathicity of the peptides might not be related to whether it is more likely to be identi?ed or not. The number of identi?ed peptides for a protein might be used to estimate its loading amount under the same sample background. Moreover, in this study the identi?ed peptides present three types of redundancy, namely iden- ti?cation, charge, and sequence redundancy, which may repress low abundance protein identi?cation.
In this study we systematically analyzed the elution condition of tryptic peptides and the characteristics of identi?ed peptides in reverse phase liquid chromatogra- phy and electrospray tandem mass spectrometry (RPLC-MS/MS) analysis. Fol- lowing protein digestion with trypsin, the peptide mixture was analyzed by on-line RPLC-MS/MS. Bovine serum albumin (BSA) was used to optimize acetonitrile (ACN) elution gradient for tryptic peptides, and Cytochrome C was used to retest the gradient and the sensitivity of LC-MS/MS. The characteristics of identi?ed peptides were also analyzed. In our experiments, the suitable ACN gradient is 5% to 30% for tryptic peptide elution and the sensitivity of LC-MS/MS is 50 fmol. Analysis of the tryptic peptides demonstrated that longer (more than 10 amino acids) and multi-charge state (+2, +3) peptides are likely to be identi?ed, and the hydropathicity of the peptides might not be related to whether it is more likely to be identi?ed or not. The number of identi?ed peptides for a protein might be used to estimate its loading amount under the same sample background. Moreover, in this study the identi?ed peptides present three types of redundancy, namely iden- ti?cation, charge, and sequence redundancy, which may repress low abundance protein identi?cation.
基金
This work was partially supported by grants fromthe National Basic Research Program (973) of China(2004CB520804), and National Natural Science Foun-dation of China (No. 30270657, 30230150, 3037030).
