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

4-Hydroxy-2-nonenal (HNE)-modified proteins are closely associated with cellular functions and diseases, so qualitative and quantitative analysis of HNE-modified proteins is very necessary in order to further understand their structures and molecular functions. In this study, we described a six-plex isobaric labeling affinity purification (SiLAP) method based on the interaction of aminoxyTMT six-plex and anti-TMT antibody resin to identify and quantify the HNE modifications simultaneously. The labeling efficiency, ionization efficiency of the aminoxyTMT-tagged peptides, and reliability of the quantification method were investigated in detail. The mass tags were labeled on the modification sites, which could also significantly increase the ionization efficiency, contributing to site-specific identification and quantification of HNE peptides. The SiLAP strategy possessed high sensitivity, accuracy, and good reproducibility to qualitatively and quantitatively analyze HNE-modified proteins/peptides, which could be used to analyze both endogenously and exogenously modified proteins. Using the SiLAP strategy, 2257 HNE-modified peptides mapping 1121 proteins were collectively quantified, which was the largest data set of HNE-modified proteins with detailed modification sites, and 101 proteins were found to be differentially modified by HNE in six liver cell lines. At the same time, 33 endogenously HNE-modified peptides mapping 33 proteins were identified with modification sites.