Journal
JOURNAL OF PROTEOME RESEARCH
Volume 17, Issue 6, Pages 2226-2236Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.jproteome.8b00217
Keywords
sample preparation; multi-notch; SPS; synchronous precursor selection; tandem mass tag; Orbitrap Fusion Lumos; phosphoproteome
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Funding
- NIH/NIDDK [K01 DK098285, GM97645]
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Mass spectrometry (MS) coupled toisobaric labeling has developed rapidly into a powerful strategy for high-throughput protein quantification. Sample multiplexing and exceptional sensitivity allow for the quantification of tens of thousands of peptides and, by inference, thousands of proteins from multiple samples in a single MS experiment. Accurate quantification demands a consistent and robust sample-preparation strategy. Here, we present a detailed workflow for SPS-MS3-based quantitative abundance profiling of tandem mass tag (TMT)-labeled proteins and phosphopeptides that we have named the streamlined (SL)-TMT protocol. We describe a universally applicable strategy that requires minimal individual sample processing and permits the seamless addition of a phosphopeptide enrichment step (mini-phos) with little deviation from the deep proteome analysis. To showcase our workflow, we profile the proteome of wild-type Saccharomyces cerevisiae yeast grown with either glucose or pyruvate as the carbon source. Here, we have established a streamlined TMT protocol that enables deep proteome and medium-scale phosphoproteome analysis.
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