Screening of Chemical Modifications in Human Skin Keratins by Mass Spectrometry-Based Proteomic Analysis via Noninvasive Sampling and On-Tape Digestion

Proteins
are continuously exposed to diverse chemical stresses,
and the resulting chemical modifications can provide significant information
on biological events. Keratins are the main constituent of human skin
and are the major target proteins of various chemical modifications.
We have previously developed a mass spectrometry-based noninvasive
proteomic methodology to screen oxidative modifications in human skin
keratins. We have improved this methodology in terms of sample preparation
time and amino acid sequence coverage using an on-tape digestion method.
After sampling by tape stripping, skin proteins on the tape were subjected
to reduction/alkylation, followed by trypsin digestion without a presolubilization
step using detergents. To screen chemical modifications in keratins,
target modifications and tryptic target peptides carrying the modification
sites were determined from in vitro experiments with
major reactive chemical species (4-hydroxy-2­(E)-nonenal
(HNE), 4-oxo-2­(E)-nonenal, glucose, methylglyoxal,
peroxynitrite, and hydrogen peroxide). The developed method was used
to screen target modifications in controls and patients with a swollen
red rash. Basal levels of lipid-derived modification, oxidation, nitration,
and glycation in keratins were detected in controls. Principal component
analysis based on the relative chemical modification resulted in a
clear classification of both groups within a 95% confidence interval.
Lipid-derived HNE modification increased most significantly in the
patient group. This methodology can be easily applied to patients
with other diseases, and the target modifications can be used as biomarkers
of certain physiological conditions.