Background

Skin, the largest barrier organ in the body, has a tough and pliable structure to adapt to external conditions by quickly repairing mechanical, chemical, and biological injuries. In mammals, the skin consists of several distinct layers: the epidermal, dermal, and subcutaneous fat layers. The epidermis is the outermost layer, with stratified cell layers maintained by keratinocytes including stem cells and an abundance of mature cells (Gonzales and Fuchs, 2017). The basal layers of the epidermis, which express the keratins KRT5 and KRT14, are the location of undifferentiated proliferative epidermal stem cells (Blanpain and Fuchs, 2006). The progenitor cells replenish basal layers and differentiate into mature keratinocytes in the granulosum and spinosum layers, which express KRT1, KRT10, and involucrin. Stratum corneum, the outermost layer, consisting of terminally differentiated and dead cells, acts as a scaffold for the lipid bilayers that comprise the epidermal barrier on the skin surface (Fuchs, 2007; Koster and Roop, 2007). Keratins, which constitute the cytoskeleton of epithelial cells, are highly expressed in the epidermis, especially in the stratum corneum (Li et al., 2022). In the epidermis, the transition of keratinocytes from the proliferative basal to the supra-basal cell layer during terminal differentiation and keratinization is characterized by keratin expression transition from basal cell keratins (KRT5, KRT14, and KRT15) to supra-basal cell keratins (KRT1 and KRT10) (Moll et al., 2008).The dermis provides nourishment and support for the skin and contains an abundance of extracellular matrix (ECM) proteins including collagens, glycosaminoglycans, hyaluronic acid, fibronectin, elastin, and laminin (Nyström and Bruckner-Tuderman, 2019). Basement membrane, a specialized layer of ECM proteins connecting the dermis and epidermis, serves as an important microenvironment for basal stem cells; understanding its composition is crucial to the study of basal stem cell fate and function. However, there is a lack of detailed information about the molecular composition and regulatory function of specialized proteins localized in different skin layers that are difficult to separate, especially the basement membrane zone, during the dynamic processes of skin development, homeostasis, and regeneration for wound healing (Dengjel et al., 2020; Dyring-Andersen et al., 2020).

As described above, the skin is a complex tissue composed of heterogeneous cell types with different spatial distributions. Studying the unique physiological and pathological functions of different spatially distributed cell types is essential for understanding the molecular characteristics of human skin. Laser capture microdissection (LCM) is a powerful technique for separating target cell populations with extremely high microscopic precision, thus providing a perfect solution to the problem of skin tissue heterogeneity. Here, we describe a protocol for the isolation of different skin layers by LCM for spatial proteomics research (Li et al., 2022). Using a combination of previously developed tissue engineering decellularization methods, dedicated to the removal of epidermis and separation of basement membrane (Leng et al., 2020, Liu et al., 2020), LCM, and mass spectrometry (MS), we isolated proteins from six skin layers (stratum corneum, stratum granular-spinous, basal layer, basement membrane, superficial dermis, and deep dermis) and constructed a stratified developmental lineage proteome map of human skin. By obtaining different skin samples, the protocol enables the analysis of spatial protein expression in normal and disease-specific skin tissues, which is important for understanding the pathogenesis of different skin diseases and discovering potential therapeutic targets.