[Identification and relative quantification of peptides by capillary reversed-phase liquid chromatography-tandem mass...

A method for the identification and relative quantification of peptides by capillary liquid chromatography ion-trap tandem mass spectrometry (LC-IT-MS/MS) was established. At first, the peptides were automatically identified by correlating the tandem mass spectra with the peptide sequences from a database. After the quantitative information of peptide ions were extracted from the full-scan MS according to the results of database searching, the peak intensities of the identified peptide ions with different charge states were summed together to define the total intensity of the peptide. Then, the peak intensities of the same peptide in the replicate analysis of the same sample were averaged and assigned as the abundance of the peptide. Finally, the abundances of the common peptide in the analysis of different samples were compared. This approach relied on the analytical reproducibility and linearity of signal versus molecular concentration. As a measure of the analytical reproducibility for tryptic peptides sampling, the median of the relative standard deviation of 35% was determined for 50 common peptides from 3 replicate analysis of 200 fmol bovine serum albumin (BSA) digest. The method was further illustrated using digested mixtures of BSA and myoglobin as follows. The BSA digest was gradually diluted while the myoglobin digest was present in the mixtures at constant level. This study revealed that the abundance of the variable BSA peptides increased linearly (trend line r2 > 0.97) with increasing amount from 10 to 1 000 fmol, while the abundances of the constant peptides from myoglobin remained approximately the same. In the present method, chemical derivatization steps are not needed to create an internal standard, as in isotope-coded affinity tag or similar methods. This method provides an alternative approach for differential analysis of peptides in biological samples.