Mass spectrometric investigation and determination of proteome composition of human skin tissues ablated using picosecond infrared laser (PIRL) in a ‘layer by layer’ approach

Abstract

Skin anatomy, its composition of abundant proteins, and the functions of these proteins are greatly established and explored. The proteome including lower abundant proteins in each type of cell layers of skin – particularly in the five different cell layers of the epidermis - are not accurately defined. The main goal of this study was the investigation of human skin proteomes in all cell layers and determination of protein composition layer-by-layer in the epidermis. Therefore, a proteomic approach was undertaken after ablating the skin tissue layer-by-layer by using an innovative, homogenization method called DIVE (desorption by impulsive vibrational excitation) using PIRL (picosecond infra-red laser). This method is based on absorption of laser energy that induces an excitation of intramolecular vibrational modes of water molecules in the cell at 3 μm Wavelength and leads to blast biomolecules out of tissues on a picosecond timescale in the gas phase. This laser ablates tissues with minimal or no damage because of its ultrafast energy transfer that cannot be converted into thermal energy or in shock waves during this short time. Another aim was to find out if PIRL can replace the conventional, mechanical homogenization methods such as disrupting tissues using bead milling through high-speed shaking with stainless steel beads (in this study termed as: TissueLyser) or grinding/milling of tissues using a mortar and pestle plus liquid nitrogen (in this study termed as grinder & LN2). Due to the need of generating a protein marker library for each main domains of the human skin tissue: epidermis and dermis, both layers had to be separated by enzymatical treatment using dispase II. The separated epidermis and dermis tissues were homogenized mechanically with either TissueLyser or with grinder & LN2 as well as with the novel homogenization method PIRL-DIVE. The proteins were analyzed by tandem mass spectrometry (MS/MS) in a bottom-up approach. With the help of existing databases “The human Protein Atlas”, protein markers were verified and categorized. When comparing the number of proteins identified in conventional homogenization methods to those identified in PIRL, the highest number of dermal proteins and the best reproducibility and protein recovery was observed with the PIRL approach. In terms of mass spectrometric proteome identification with classical methods averagely 550 proteins could be identified in dermal tissues whilst with PIRL, about 900 proteins were identified. In the case of epidermal tissues, the protein yields in both conventional, mechanical as well as in PIRL homogenization techniques were quite similar, whereas the reproducibility rate of proteins identified in PIRL-ablated epidermal tissues was higher than in conventional methods. Not forgetting, the contamination risk was reduced by PIRL because of an ultrafast homogenization and less sample preparation steps. Furthermore, the goal of the study was achieved by ablating the human skin tissue layer by layer (approximately 15 μm ablation depth per ablated layer) and determining the protein composition in each ablated cell layer using tandem mass spectrometry.