Ultrafast and Reproducible Proteomics from Small Amounts of Heart Tissue Enabled by Azo and timsTOF Pro

Global bottom-up mass spectrometry
(MS)-based proteomics is widely
used for protein identification and quantification to achieve a comprehensive
understanding of the composition, structure, and function of the proteome.
However, traditional sample preparation methods are time-consuming,
typically including overnight tryptic digestion, extensive sample
cleanup to remove MS-incompatible surfactants, and offline sample
fractionation to reduce proteome complexity prior to online liquid
chromatography–tandem mass spectrometry (LC-MS/MS) analysis.
Thus, there is a need for a fast, robust, and reproducible method
for protein identification and quantification from complex proteomes.
Herein, we developed an ultrafast bottom-up proteomics method enabled
by Azo, a photocleavable, MS-compatible surfactant that effectively
solubilizes proteins and promotes rapid tryptic digestion, combined
with the Bruker timsTOF Pro, which enables deeper proteome coverage
through trapped ion mobility spectrometry (TIMS) and parallel accumulation–serial
fragmentation (PASEF) of peptides. We applied this method to analyze
the complex human cardiac proteome and identified nearly 4000 protein
groups from as little as 1 mg of human heart tissue in a single one-dimensional
LC-TIMS-MS/MS run with high reproducibility. Overall, we anticipate
this ultrafast, robust, and reproducible bottom-up method empowered
by both Azo and the timsTOF Pro will be generally applicable and greatly
accelerate the throughput of large-scale quantitative proteomic studies.
Raw data are available via the MassIVE repository with identifier
MSV000087476.