Quantitative Consequences of Protein Carriers in Immunopeptidomics and Tyrosine Phosphorylation MS2 Analyses

• Published in: Molecular & cellular proteomics Vol. 20; p. 100104
• Main Authors: Stopfer, Lauren E., Conage-Pough, Jason E., White, Forest M.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01.01.2021; American Society for Biochemistry and Molecular Biology
• Subjects: BOOST channel; immunopeptidomics; MHC class I; phosphoproteomics; phosphotyrosine; protein carrier; quantification; phosphoproteomics; DDA; SCP; RT; BOOST channel; DMSO; PV; pTyr; MS3; EGFR; phosphotyrosine; nCE; IFN-γ; protein carrier; AGC; PBS; BCA; HipMHC; quantification; CDK4/6; EGF; MS; MHC class I; HCD; IT; PSM; pMHC; FAIMS; PCA; CV; PC; PD; SP3; TMT; immunopeptidomics
• ISSN: 1535-9476; 1535-9484; 1535-9484
• DOI: 10.1016/j.mcpro.2021.100104

Utilizing a protein carrier in combination with isobaric labeling to “boost” the signal of other low-level samples in multiplexed analyses has emerged as an attractive strategy to enhance data quantity while minimizing protein input in mass spectrometry analyses. Recent applications of this approach include pMHC profiling and tyrosine phosphoproteomics, two applications that are often limited by large sample requirements. While including a protein carrier has been shown to increase the number of identifiable peptides in both applications, the impact of a protein carrier on quantitative accuracy remains to be thoroughly explored, particularly in relevant biological contexts where samples exhibit dynamic changes in abundance across peptides. Here, we describe two sets of analyses comparing MS2-based quantitation using a 20× protein carrier in pMHC analyses and a high (~100×) and low (~9×) protein carrier in pTyr analyses, using CDK4/6 inhibitors and EGF stimulation to drive dynamic changes in the immunopeptidome and phosphoproteome, respectively. In both applications, inclusion of a protein carrier resulted in an increased number of MHC peptide or phosphopeptide identifications, as expected. At the same time, quantitative accuracy was adversely affected by the presence of the protein carrier, altering interpretation of the underlying biological response to perturbation. Moreover, for tyrosine phosphoproteomics, the presence of high levels of protein carrier led to a large number of missing values for endogenous phosphopeptides, leading to fewer quantifiable peptides relative to the “no-boost” condition. These data highlight the unique limitations and future experimental considerations for both analysis types and provide a framework for assessing quantitative accuracy in protein carrier experiments moving forward. [Display omitted] •A carrier proteome may enhance detection of low abundance pMHC and pTyr peptides.•A carrier boosted IDs in pMHC analyses; quantitation suffered from ratio compression.•A 9× carrier provided higher quantitative accuracy over a 100× carrier in pTyr analyses but offered minimal benefit in data quantity.•Experimental design should be rigorously evaluated to ensure quantitative accuracy. Use of a protein carrier offers the opportunity to increase data quantity while minimizing sample input requirements, an attractive strategy for tyrosine phosphorylation and immunopeptidomics analyses. Still, the quantitative consequences of including a protein carrier in pMHC and pTyr analyses, particularly in a biological context, remain poorly understood. Here, a comparison of experiments with and without a carrier proteome reveals critical limitations, suggesting that experimental design of carrier experiments should be carefully assessed to avoid misinterpretation of data.