Active Instrument Engagement Combined with a Real-Time Database Search for Improved Performance of Sample Multiplexing Workflows

Quantitative proteomics
employing isobaric reagents has been established
as a powerful tool for biological discovery. Current workflows often
utilize a dedicated quantitative spectrum to improve quantitative
accuracy and precision. A consequence of this approach is a dramatic
reduction in the spectral acquisition rate, which necessitates the
use of additional instrument time to achieve comprehensive proteomic
depth. This work assesses the performance and benefits of online and
real-time spectral identification in quantitative multiplexed workflows.
A Real-Time Search (RTS) algorithm was implemented to identify fragment
spectra within milliseconds as they are acquired using a probabilistic
score and to trigger quantitative spectra only upon confident peptide
identification. The RTS-MS3 was benchmarked against standard
workflows using a complex two-proteome model of interference and a
targeted 10-plex comparison of kinase abundance profiles. Applying
the RTS-MS3 method provided the comprehensive characterization
of a 10-plex proteome in 50% less acquisition time. These data indicate
that the RTS-MS3 approach provides dramatic performance
improvements for quantitative multiplexed experiments.