Chemical Modifications Induced by Phthalic Anhydride, a Respiratory Sensitizer, in Reconstructed Human Epidermis: A Combined HRMAS NMR and LC-MS/MS Proteomic Approach

Chemical skin and respiratory allergies are becoming a major health
problem. To date our knowledge on the process of protein haptenation
is still limited and mainly derived from studies performed in solution
using model nucleophiles. In order to better understand chemical interactions
between chemical allergens and the skin, we have investigated the
reactivity of phthalic anhydride 1 (PA), a chemical respiratory
sensitizer, toward reconstructed human epidermis (RHE). This study
was performed using a new approach combining HRMAS NMR to investigate
the in situ chemical reactivity and LC-MS/MS to identify modified
epidermal proteins. In RHE, the reaction of PA appeared to be quite
fast and the major product formed was phthalic acid. Two amide type
adducts on lysine residues were observed and after 8h of incubation,
we also observed the formation of an imide type cyclized adducts with
lysine. In parallel, RHE samples topically exposed to phthalic anhydride
(13C)-1 were analyzed using the shotgun proteomics method.
Thus, 948 different proteins were extracted and identified, 135 of
which being modified by PA, i.e., 14.2% of the extracted proteome.
A total of 211 amino acids were modified by PA and validated by fragmentation
spectra. We thus identified 154 modified lysines, 22 modified histidines,
30 modified tyrosines, and 5 modified arginines. The rate of modified
residues, as a proportion of the total number of modifiable nucleophilic
residues in RHE, was rather low (1%). At the protein level, modified
proteins were mainly type I and type II keratins and other proteins
which are abundant in the epidermis such as protein S100A, Caspase
14, annexin A2, serpin B3, fatty-acid binding protein 5, histone H2,
H3, H4, etc. However, the most modified protein, mainly on histidine
residues, was filaggrin, a protein that is of low abundance (0.0266
mol %) and rich in histidine.