Abstract
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.
Highlights
A carrier proteome may enhance detection of low abundance peptide major histocompatibility complex (pMHC) and tyrosine phosphorylation (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
To interrogate the impact of including a carrier proteome on pMHC identification and quantitation, we prepared a set of six cell-line-derived replicate samples comprised of 1 × 106 cells per channel for the analysis without a protein carrier (“noboost”), and a parallel experiment using 50% fewer cells per sample (5 × 105 cells) for the “MHC-boost” analysis (Fig. 1, A and B)
We utilized a panel of six synthetic, heavy-isotope labeled pMHCs, which were titrated into cell lysates prior to pMHC isolation to generate an internal standard curve against a consistent background immunopeptidome, as previously described [7]
Summary
In Brief 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. 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. 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. We define existing limitations for MS2-based analyses using protein carriers and highlight areas for future exploration that may enhance data quality through altered experimental design or acquisition framework
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