Selective Identification and Site-Specific Quantification of 4-Hydroxy-2-nonenal-Modified Proteins

Area of Science:

• Proteomics
• Biochemistry
• Cellular Biology

Background:

• 4-Hydroxy-2-nonenal (HNE)-modified proteins are crucial in cellular functions and disease.
• Accurate qualitative and quantitative analysis of HNE modifications is essential for understanding protein structure and function.

Purpose of the Study:

• To develop and validate a novel method for simultaneous identification and quantification of HNE-modified proteins.
• To establish a comprehensive dataset of HNE-modified proteins and their modification sites.

Main Methods:

• A six-plex isobaric labeling affinity purification (SiLAP) method was developed using aminoxyTMT six-plex and anti-TMT antibody resin.
• The method was optimized for labeling efficiency, ionization efficiency, and quantification reliability.
• SiLAP was applied to analyze both endogenous and exogenous HNE modifications.

Main Results:

• The SiLAP strategy demonstrated high sensitivity, accuracy, and reproducibility.
• A dataset of 2257 HNE-modified peptides from 1121 proteins was quantified, representing the largest such dataset with detailed modification sites.
• 101 proteins showed differential HNE modification across six liver cell lines, and 33 endogenous HNE-modified peptides were identified.

Conclusions:

• The SiLAP method is a powerful tool for comprehensive qualitative and quantitative analysis of HNE-modified proteins.
• This approach significantly advances the study of HNE modifications in cellular processes and disease pathogenesis.
• The generated dataset provides valuable insights into the biological roles of HNE modification.