Global Extraction from Parallel Reaction Monitoring to Quantify Background Peptides for Improved Normalization and Quality Control in Targeted Proteomics

Mass spectrometry-based targeted
proteomics employs heavy isotope-labeled
proteins or peptides as standards to improve accuracy and precision.
The input sample amount is often determined by the total quantity
of endogenous proteins or peptides, as defined by spectrophotometric
assays, before the heavy-isotope standards are spiked into the samples.
Errors in spectrophotometric measurements, which may be due to low
sensitivity or chemical or biological interference, have a direct
impact on the quantitative mass spectrometry results. Currently used
targeted proteomics workflows cannot identify or correct deviations
that arise from differences in the input sample amount. We have developed
a workflow, global extraction from parallel reaction monitoring (PRM),
to identify and quantify thousands of background peptides that are
inherently acquired by PRM experiments. These background peptides
were used to identify differences in the input sample amount and to
reduce this variance by intensity-based, post-acquisition normalization.
This approach was then applied to a xenograft study to improve the
quantification of human proteins in the presence of mouse tissue contamination.
In addition, these background peptides also provided a direct source
of quality control metrics related to sample handling and preparation.