Automated Workflow for Large-Scale Selected Reaction Monitoring Experiments

Targeted proteomics allows researchers to study proteins
of interest
without being drowned in data from other, less interesting proteins
or from redundant or uninformative peptides. While the technique is
mostly used for smaller, focused studies, there are several reasons
to conduct larger targeted experiments. Automated, highly robust software
becomes more important in such experiments. In addition, larger experiments
are carried out over longer periods of time, requiring strategies
to handle the sometimes large shift in retention time often observed.
We present a complete proof-of-principle software stack that automates
most aspects of selected reaction monitoring workflows, a targeted
proteomics technology. The software allows experiments to be easily
designed and carried out. The steps automated are the generation of
assays, generation of mass spectrometry driver files and methods files,
and the import and analysis of the data. All data are normalized to
a common retention time scale, the data are then scored using a novel
score model, and the error is subsequently estimated. We also show
that selected reaction monitoring can be used for label-free quantification.
All data generated are stored in a relational database, and the growing
resource further facilitates the design of new experiments. We apply
the technology to a large-scale experiment studying how Streptococcus pyogenes remodels its proteome
under stimulation of human plasma.