Phosphoproteome Profiling Using an Isobaric Carrier without the Need for Phosphoenrichment.

Area of Science:

• Proteomics
• Cellular Signaling
• Biochemistry

Background:

• Phosphorylation is vital for cellular functions like growth and apoptosis.
• Low phosphopeptide stoichiometry necessitates enrichment for LC-MS/MS analysis.
• Current quantitative phosphoproteomics requires substantial sample amounts.

Purpose of the Study:

• To develop a reproducible strategy for increasing phosphoproteomic signals from limited samples.
• To enable comprehensive phosphoproteome analysis without a phosphoenrichment step.
• To facilitate quantitative phosphoproteomics with reduced sample input.

Main Methods:

• Exploited isobaric labeling multiplexing for merged sample signals.
• Utilized enriched phosphopeptides as a carrier to boost trace amounts.
• Quantified phosphopeptides using reporter ion intensity from isobaric tags.

Main Results:

• Quantified over 1400 phosphopeptides from just 250 ng of tryptic peptides.
• Successfully distinguished three lung cancer cell lines using quantitative phosphoproteomic data.
• Identified drug-induced changes in the phosphoproteome.

Conclusions:

• The developed strategy enhances phosphoproteomic signals from small samples effectively.
• This method reduces sample requirements for quantitative phosphoproteomics.
• The approach is applicable for cell line discrimination and drug effect analysis.