Highly Selective and Large Scale Mass Spectrometric Analysis of 4‑Hydroxynonenal Modification via Fluorous Derivatization and Fluorous Solid-Phase Extraction

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.