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

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Chemical Modifications Induced by Phthalic Anhydride, a Respiratory Sensitizer, in Reconstructed Human Epidermis: A Combined HRMAS NMR and LC-MS/MS Proteomic Approach
Chemical Research in Toxicology ( IF 3.7 ) Pub Date : 2021-08-09 , DOI: 10.1021/acs.chemrestox.1c00172
Minh-Thuong Khong ₁ , Valérie Berl ₁ , Lauriane Kuhn ₂ , Philippe Hammann ₂ , Jean-Pierre Lepoittevin ₁

Affiliation

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.

中文翻译：

邻苯二甲酸酐（一种呼吸敏化剂）在重建的人类表皮中引起的化学修饰：一种结合 HRMAS NMR 和 LC-MS/MS 蛋白质组学方法

化学性皮肤和呼吸道过敏正在成为一个主要的健康问题。迄今为止，我们对蛋白质半抗原化过程的了解仍然有限，主要来自使用模型亲核试剂在溶液中进行的研究。为了更好地了解化学过敏原与皮肤之间的化学相互作用，我们研究了邻苯二甲酸酐的反应性1(PA)，一种化学呼吸敏化剂，用于重建人类表皮 (RHE)。这项研究是使用一种新方法结合 HRMAS NMR 来研究原位化学反应性和 LC-MS/MS 来识别修饰的表皮蛋白。在 RHE 中，PA 的反应似乎非常快，形成的主要产物是邻苯二甲酸。观察到赖氨酸残基上的两种酰胺型加合物，孵育 8 小时后，我们还观察到与赖氨酸形成酰亚胺型环化加合物。同时，RHE 样品局部暴露于邻苯二甲酸酐 ( ¹³C)-1 使用鸟枪蛋白质组学方法进行分析。因此，提取和鉴定了 948 种不同的蛋白质，其中 135 种被 PA 修饰，即提取的蛋白质组的 14.2%。共有 211 个氨基酸被 PA 修饰并通过碎片光谱验证。因此，我们鉴定了 154 个修饰的赖氨酸、22 个修饰的组氨酸、30 个修饰的酪氨酸和 5 个修饰的精氨酸。修饰残基占 RHE 中可修饰亲核残基总数的比例相当低 (1%)。在蛋白质水平上，修饰的蛋白质主要是 I 型和 II 型角蛋白以及表皮中丰富的其他蛋白质，如蛋白质 S100A、Caspase 14、膜联蛋白 A2、丝氨酸蛋白酶抑制剂 B3、脂肪酸结合蛋白 5、组蛋白 H2、H3、 H4等。然而，修饰最多的蛋白质，主要是组氨酸残基，是丝聚蛋白，

更新日期：2021-09-20

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