Expediting SRM Assay Development for Large-Scale Targeted Proteomics Experiments

Because
of its high sensitivity and specificity, selected reaction
monitoring (SRM)-based targeted proteomics has become increasingly
popular for biological and translational applications. Selection of
optimal transitions and optimization of collision energy (CE) are
important assay development steps for achieving sensitive detection
and accurate quantification; however, these steps can be labor-intensive,
especially for large-scale applications. Herein, we explored several
options for accelerating SRM assay development evaluated in the context
of a relatively large set of 215 synthetic peptide targets. We first
showed that HCD fragmentation is very similar to that of CID in triple
quadrupole (QQQ) instrumentation and that by selection of the top
6 y fragment ions from HCD spectra, >86% of the top transitions
optimized
from direct infusion with QQQ instrumentation are covered. We also
demonstrated that the CE calculated by existing prediction tools was
less accurate for 3+ precursors and that a significant increase in
intensity for transitions could be obtained using a new CE prediction
equation constructed from the present experimental data. Overall,
our study illustrated the feasibility of expediting the development
of larger numbers of high-sensitivity SRM assays through automation
of transition selection and accurate prediction of optimal CE to improve
both SRM throughput and measurement quality.