title: Highly Selective and Large Scale Mass Spectrometric Analysis of 4-Hydroxynonenal Modification via Fluorous Derivatization and Fluorous Solid-Phase Extraction.
abstract: Modification of proteins with 4-hydroxynonenal (HNE) is known to alter the function of proteins and regulate the associated biological processes in eukaryotic cells. The development of mass spectrometry (MS) makes high-throughput analysis of HNE modification accessible. However, the identification of HNE modification is still hampered by the low frequency of this modification. Therefore, only a limited number of HNE modification sites have been identified. The enrichment of HNE-modified peptides is critical for the MS analysis of this modification because of its low abundance. Herein, we explored a novel strategy for specifically extracting the HNE-modified peptides using fluorous derivatization through oxime click chemistry combined with following fluorous solid-phase extraction (FSPE). This oxime click chemistry-based derivatization is highly efficient (with a yield of almost 100%) and fast (30 min). Because of the hydrophobicity of the fluorous tag, the signal of fluorous-derivatized HNE-modified peptides was greatly enhanced, making the detection of HNE-modified peptides sensitive. The FSPE further allowed the selective enrichment of fluorous-derivatized HNE-modified peptides from salt solutions and complex mixtures with specificity. Finally, 673 HNE modification sites (607 histidine sites, 60 cysteine sites, 5 lysine sites, and 1 arginine site) on 661 HNE-modified peptides mapped to 432 proteins were successfully identified using this novel approach, which presented the largest data set of HNE modification in MCF-7 cells. Three identified proteins were validated by Western blotting.
authors.affiliation: Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University , Shanghai 200032, P. R. China.|Department of Chemistry, Fudan University , Shanghai 200433, P. R. China.
authors.forename: Wenjuan
authors.initials: W
authors.lastname: Yuan
authors.affiliation: Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University , Shanghai 200032, P. R. China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University , Shanghai 200032, P. R. China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Xiong
authors.affiliation: Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University , Shanghai 200032, P. R. China.|Department of Chemistry, Fudan University , Shanghai 200433, P. R. China.
authors.forename: Tao
authors.initials: T
authors.lastname: Tao
authors.affiliation: Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University , Shanghai 200032, P. R. China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University , Shanghai 200032, P. R. China.
authors.forename: Jun
authors.initials: J
authors.lastname: Yao
authors.affiliation: Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University , Shanghai 200032, P. R. China.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Department of Chemistry, Fudan University , Shanghai 200433, P. R. China.
authors.forename: Guoquan
authors.initials: G
authors.lastname: Yan
authors.affiliation: Department of Chemistry, Fudan University , Shanghai 200433, P. R. China.
authors.forename: Huimin
authors.initials: H
authors.lastname: Bao
authors.affiliation: Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University , Shanghai 200032, P. R. China.|Department of Chemistry, Fudan University , Shanghai 200433, P. R. China.|Key Laboratory of Glycoconjugates Research Ministry of Public Health, Fudan University , Shanghai 200032, P. R. China.
authors.forename: Haojie
authors.identifier: 0000-0003-3477-7662
authors.initials: H
authors.lastname: Lu