Streamlined Tandem Mass Tag (SL-TMT) Protocol: An Efficient Strategy for Quantitative (Phospho)proteome Profiling Using Tandem Mass Tag-Synchronous Precursor Selection-MS3

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.