Phosphoproteome Profiling Using an Isobaric Carrier without the Need for Phosphoenrichment
datasetposted on 2022-03-03, 18:33 authored by Yumi Kwon, Seonjeong Lee, Narae Park, Shinyeong Ju, Sungho Shin, Seongjin Yoo, Hankyul Lee, Cheolju Lee
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.
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tryptic peptides preparedreporter ion intensityquantitative phosphoproteomic workflowsquantitative phosphoproteomic dataincrease trace amountsincrease phosphoproteomic signalsincluding cell growthdistinguished three typescellular signaling cascadesbiological cellular functionscomprehensive phosphoproteome analysisperform quantification usingphosphoproteome profiling usinglc – msisobaric carrier withoutphosphoproteome inducedsample withoutisobaric tagisobaric labelingwidely applicableunpurified sampletypically limitedsample requiredresults showedphosphorylated proteinsphosphoenrichment stepphosphoenrichment phosphorylationmultiplexing naturemultiple samplesmerged signallow stoichiometryidentified changesidentifiable leveldrug treatmentcrucial component250 ng