Isobaric Stable Isotope N-Phosphorylation Labeling (iSIPL) for Ultrasensitive Proteome Quantification

Area of Science:

• Proteomics
• Mass Spectrometry
• Chemical Biology

Background:

• High-throughput mass spectrometry (MS)-based quantitative proteomics relies on stable isotope chemical labeling.
• Existing labeling methods lack the high sensitivity required for biological and clinical applications.

Purpose of the Study:

• To develop a novel isobaric stable isotope N-phosphorylation labeling (iSIPL) strategy for enhanced quantitative proteome analysis.
• To improve sensitivity and accuracy in mass spectrometry-based proteomics, especially for limited sample quantities.

Main Methods:

• Selective labeling of tryptic peptides using the iSIPL tag.
• Generation of novel reporter ions with high intensities under lower collision energies via a phosphoramidate P-N bond.
• Coupling iSIPL with affinity purification and mass spectrometry for dynamic interactome analysis.

Main Results:

• The iSIPL strategy enables sensitive and accurate peptide sequencing and quantitative analysis, even with limited sample amounts.
• Application of iSIPL in studying cyclin dependent kinase 9 (CDK9) interactomes during HIV-1 provirus transactivation revealed a significant decrease in CDK9-PARP13 interaction upon Tat-induced transcription.
• Demonstrated the effectiveness of iSIPL in dynamic analysis of in vivo protein-protein interactions.

Conclusions:

• The developed iSIPL strategy offers high sensitivity and accuracy for quantitative proteomics.
• iSIPL is effective for dynamic analysis of protein-protein interactions, as shown in HIV-1 transcription studies.
• The iSIPL strategy has the potential to advance large-scale quantitative proteomics by enhancing multiplexing capabilities.