Strain-Specific Peptide (SSP) Interference Reference Sample: A Genetically Encoded Quality Control for Isobaric Tagging Strategies

Isobaric
tag-based sample multiplexing strategies are extensively
used for global protein abundance profiling. However, such analyses
are often confounded by ratio compression resulting from the co-isolation,
co-fragmentation, and co-quantification of co-eluting peptides, termed
“interference.” Recent analytical strategies incorporating
ion mobility and real-time database searching have helped to alleviate
interference, yet further assessment is needed. Here, we present the
strain-specific peptide (SSP) interference reference sample, a tandem
mass tag (TMT)­pro-labeled quality control that leverages the genetic
variation in the proteomes of eight phylogenetically divergent mouse
strains. Typically, a peptide with a missense mutation has a different
mass and retention time than the reference or native peptide. TMT
reporter ion signal for the native peptide in strains that encode
the mutant peptide suggests interference which can be quantified and
assessed using the interference-free index (IFI). We introduce the
SSP by investigating interference in three common data acquisition
methods and by showcasing improvements in the IFI when using ion mobility-based
gas-phase fractionation. In addition, we provide a user-friendly,
online viewer to visualize the data and streamline the calculation
of the IFI. The SSP will aid in developing and optimizing isobaric
tag-based experiments.