Phosphoproteome Profiling Using an Isobaric Carrier without the Need for Phosphoenrichment

Phosphorylation
is a crucial component of cellular signaling cascades.
It controls a variety of biological cellular functions, including
cell growth and apoptosis. Owing to the low stoichiometry of phosphorylated
proteins, the enrichment of phosphopeptides prior to LC–MS/MS
is necessary for comprehensive phosphoproteome analysis, and quantitative
phosphoproteomic workflows are typically limited by the amount of
sample required. To address this issue, we developed an easy-to-establish,
widely applicable, and reproducible strategy to increase phosphoproteomic
signals from a small amount of sample without a phosphoenrichment
step. By exploiting the multiplexing nature of isobaric labeling to
generate a merged signal from multiple samples, and using a larger
amount of enriched phosphopeptides as a carrier, we were able to increase
trace amounts of phosphopeptides in the unpurified sample to an identifiable
level and perform quantification using the reporter ion intensity
of the isobaric tag. Our results showed that >1400 phosphopeptides
were quantified from 250 ng of tryptic peptides prepared from cells.
In a proof-of-concept of our strategy, we distinguished three types
of lung cancer cell lines based on their quantitative phosphoproteomic
data and identified changes in the phosphoproteome induced by drug
treatment.