Accurate and Sensitive Quantitation of the Dynamic Heat Shock Proteome Using Tandem Mass Tags

Cells respond to environmental perturbations
and insults through
modulating protein abundance and function. However, the majority of
studies have focused on changes in RNA abundance because quantitative
transcriptomics has historically been more facile than quantitative
proteomics. Modern Orbitrap mass spectrometers now provide sensitive
and deep proteome coverage, allowing direct, global quantification
of not only protein abundance but also post-translational modifications
(PTMs) that regulate protein activity. We implemented and validated
using the well-characterized heat shock response of budding yeast,
a tandem mass tagging (TMT), triple-stage mass spectrometry (MS3) strategy to measure global changes in the proteome during
the yeast heat shock response over nine time points. We report that
basic-pH, ultra-high performance liquid chromatography (UPLC) fractionation
of tryptic peptides yields superfractions of minimal redundancy, a
crucial requirement for deep coverage and quantification by subsequent
LC–MS3. We quantified 2275 proteins across three
biological replicates and found that differential expression peaked
near 90 min following heat shock (with 868 differentially expressed
proteins at 5% false discovery rate). The sensitivity of the approach
also allowed us to detect changes in the relative abundance of ubiquitination
and phosphorylation PTMs over time. Remarkably, relative quantification
of post-translationally modified peptides revealed striking evidence
of regulation of the heat shock response by protein PTMs. These data
demonstrate that the high precision of TMT-MS3 enables
peptide-level quantification of samples, which can reveal important
regulation of protein abundance and regulatory PTMs under various
experimental conditions.