Effect of Collision Energy Optimization on the Measurement of Peptides by Selected Reaction Monitoring (SRM) Mass Spectrometry

摘要

In these experiments we have used a fully automated workflow, implemented in version 0.6 of the open source software Skyline, to evaluate the effect of optimizing CE values across multiple instrument platforms. By comparing peak areas at different CE values for different peptide sequences and precursor charge-states we have quantified the improvement in sensitivity obtained by optimizing each transition for each peptide individually compared to using a simple linear equation to predict CE based on the peptide precursor mass. We have derived optimized linear equations for predicting CE values that are different from the default equations recommended by the manufacturer or reported in the literature. These new equations for predicting CE are available in the latest release of Skyline. With well optimized linear equations, we have shown losses of 8.4percent of total peak area on average compared with fully optimizing each transition. Understanding the variance in the transition peak area to CE relationship is critical to understanding how any peak area optimization using CE will perform. We found that low concentrations for certain peptides yielded measurements that were too noisy to support CE optimization. A similar effect occurs when using an optimal CE value determined with a standard at high abundance to measure a low abundance species in a real sample. Even if the measurement is made with the optimal CE, the improvement in sensitivity over a less optimal CE will be indistinguishable at low intensity because the variance from the measurement shot noise will overwhelm the small improvement in peak area.