Sara Ceccacci (Paris / FR), Lucile Marchal (Paris / FR), Hélène Ragot (Paris / FR), Janice Schwartz (Salt Lake City, UT / US), Alain Hovnanian (Paris / FR), Ida Chiara Guerrera (Paris / FR)
Pachyonychia congenita (PC) is a rare skin disorder caused by heterozygous mutations in keratin genes (KRT6A, KRT6B, KRT6C, KRT16 or KRT17). The most severe clinical feature is the intense pain associated with palmoplantar keratoderma. To date, there are no satisfactory therapeutic options. An unbiased mass spectrometry (MS)-based proteomics analysis of patient skin biopsies could help to elucidate the elusive pathogenic mechanisms driving PC and to pinpoint new potential therapeutic targets. However, proteomic studies of skin samples present significant challenges. The most limiting step is protein extraction, as skin tissues are hard to homogenize due to the abundance of structural proteins like keratins and collagens. Furthermore, OCT-preserved skin samples also require the removal of OCT, which is incompatible with MS analysis, leading to longer extraction protocol and low protein yield. These issues are even more challenging in skin biopsies from patients with keratoderma. In our study, we optimized a high-throughput MS-based workflow for skin sample analysis.
5 mm snap-frozen skin tissues from healthy donors (controls) were divided by 4 to obtain the equivalent of 2 mm skin biopsies and then homogenized using BeatBox Tissue Kit 24x (PreOmics). 10µm slides from OCT tissues from the same controls and OCT tissues from three PC patients with KRT6A and KRT16 mutations (callus edges and adjacent non-lesional areas) were washed with PBS before BeatBox homogenization. Each sample was subjected to 96 well S-Trap plate (Protifi) digestion. 600ng of peptides were injected on an EvosepOne LC- timsTOF HT MS (Bruker) and eluted using a 30SDP method. MS data were acquired using diaPASEF and analyzed by DIA-NN v1.8.1.
We obtained the highest skin tissue proteome coverage reported to date in a single-shot MS analysis, reducing dramatically the protein extraction time (24 samples in 1 hour). We identified an average of 6,800 proteins in the snap-frozen tissue samples and 5,200 proteins in the OCT tissues from the controls. Levels of outer epidermis (KRT1 and KRT14) and dermis markers (COL3A1) were similar in snap-frozen and OCT samples, confirming that the extraction was not biased. Despite the presence of keratoderma, OCT tissues from the three PC patients led to 6,800 PG. Of these, 196 proteins were deregulated in lesional versus non-lesional PC tissues (FDR=0.05), including over 30 candidates highlighted in a previous transcriptomics study. Enrichment analysis of the 85 upregulated proteins confirmed hyperkeratinization and revealed significant alterations in lipid biosynthesis pathways, which explain the clinical benefits of some of the treatments described in case reports.
In conclusion, we propose a reproducible, miniaturized and in-depth proteomics workflow for analyzing large cohorts of both OCT-preserved and snap-frozen skin tissue samples. This enabled us to elucidate the pathways involved in the pathogenesis of Pachyonychia congenita.