Abstract
Proteins present in two mitochondria preparations were separated by 2‐D chromatography using the ProteomeLab^TM^ PF‐2D Protein Fractional System, protein fractionation in two dimensions (PF‐2D). The proteins in each first‐dimension fraction were determined by trypsinization and LC‐MS/MS. Chromatography peaks were quantified by UV detection using the “Mapping Tools” software (Beckman). The proteins present in UV detected peaks were trypsinized and identified by automated MS/MS sequencing. Relative amounts of the proteins present in the equivalent peak for each sample were assessed by comparison of the intensities of the constituent peptides and a predicted PF‐2D value was calculated from the total ion count (TIC) for each peptide. Relative quantification for ^18^O labeled peptides was performed using the ZoomQuant (v1.43b) software [1, 2]. We found that the chromatography peaks detected by UV generally contained several proteins. Using ^18^O labeling we determined that in each peak the ratios of the constituent proteins were different. When these ratios were normalized using the TIC to account for abundance, the resulting ratio corresponded to that determined by UV. The predicted value for the PF‐2D score corresponded to the observed value for each peak irrespective of the number or proteins detected.