posted on 2019-03-20, 00:00authored bySiwen Zhang, Caiyun Fang, Wenjuan Yuan, Ying Zhang, Guoquan Yan, Lei Zhang, Yi Di, Yan Cai, Haojie Lu
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.