Title

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

Document Type

Article

Publication Date

3-30-2021

Publication Title

Analytical chemistry

Keywords

JMG

JAX Source

Anal Chem 2021 Mar 30; 93(12):6241-5247

Volume

93

Issue

12

First Page

5241

Last Page

5247

ISSN

1520-6882

PMID

33735571

DOI

https://doi.org/10.1021/acs.analchem.0c05483

Abstract

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.

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