Skin Proteome Research Articles

Beyond the genome: protecting the proteome may be the key to preventing skin aging.

Skin aging is associated with a progressive decline in physiological functions, skin cancers and, ultimately, death. It may be categorized as intrinsic or extrinsic, whereby intrinsic aging is attributed to chronological and genetic factors. At the molecular level, skin aging involves changes in protein conformation and function. The skin proteome changes constantly, mainly through carbonylation; an irreversible phenomenon leading to protein accumulation as toxic aggregates that impair cellular physiology and accelerate skin aging. This review details the central role of proteostasis during skin aging and why proteome protection may be a promising approach in mitigating skin aging. A comprehensive literature review of 87 articles focusing on the proteome, proteostasis, proteotoxicity, protein carbonylation, and the impact of the damaged proteome on aging, and in particular skin aging, was conducted. Skin aging is associated with deficiencies in the repair mechanisms of DNA, transcriptional control, mitochondrial function, cell cycle control, apoptosis, cellular metabolism, changes in hormonal levels secondary to toxicity of damaged proteins, and cell-to-cell communication for tissue homeostasis, which are largely controlled by proteins. In this context, a damaged proteome that leads to the loss of proteostasis may be considered as the first step in tissue aging. There is growing evidence that a healthy proteome plays a central role in skin and in maintaining healthy tissues, thus slowing down the process of skin aging. Hence, protecting the proteome against oxidative or other damage may be an appropriate strategy to prevent and delay skin aging.

The use of probiotics and prebiotics in the restoration of aesthetic problems. What is a proteome?

A balanced microbiome protects against external environmental factors, strengthens the skin's defenses and slows the appearance of the signs of skin aging. In recent years, alternative treatments have been sought to treat various skin problems, through modification or supplementation of the microbiome, using prebiotics and probiotics. Cosmetics with probiotics and prebiotics are a rich source of proteins and vitamins and have been shown to strengthen the skin's proteome.

P28 Data-independent acquisition mass spectrometry improves spatially resolved analysis of the human skin proteome

Abstract Introduction and aims Proteomic analysis of the extracellular matrix (ECM) presents challenges because of the highly crosslinked and low-solubility nature of ECM proteins. Traditional homogenization and protein digestion approaches result in the loss of crucial information regarding protein localization and spatial relationships. To address this, spatially resolved proteomics emerges as a powerful tool for exploring heterogeneity within bulk tissues. This study aims to determine the minimum tissue volume required for comprehensive proteome coverage using data-independent acquisition mass spectrometry (DIA-MS) on skin tissue. The study focused on optimizing spatially resolved proteomic techniques to enhance depth-of-analysis while preserving spatial specificity. Methods Human abdominal skin biopsies were obtained from a single individual and subsequently cryosectioned. Histological assessment was performed through haematoxylin and eosin staining for visualization purposes. Laser-capture microdissection coupled with mass spectrometry facilitated the precise isolation of target regions. Comparative analyses were performed between data-dependent acquisition mass spectrometry (DDA-MS) and DIA-MS, with a particular emphasis on ECM proteins within the dermis. Results Our findings revealed an improvement in proteome coverage with DIA-MS compared with DDA-MS, in addition to clear scaling relationships between the depth-of-analysis and sample concentration. The Results demonstrated the superiority of DIA-MS in achieving robust and comprehensive proteomic profiles, even with minimal tissue volumes. Preliminary findings suggest the capability of DIA-MS in elucidating the complexities of the skin proteome with spatial resolution. Conclusions In conclusion, our study highlights the efficacy of DIA-MS in spatially resolved proteomics on skin tissue. The optimized approach presented here offers a reliable and efficient method for obtaining in-depth proteome information with minimal tissue requirements. These Results form the foundation for ongoing experiments, utilizing DIA-MS to advance spatially resolved proteomic analyses of human skin.

Skin proteomic screening and functional analysis of differential proteins associated with coat color in sheep (Ovis aries).

Coat color is an important characteristic and economic trait in domestic sheep. In this study, we explored the potential mechanisms and the signaling pathways involved in coat color regulation for sheep. Isobaric tags for relative and absolute quantification (iTRAQ) technology was used to catalog global protein expression profiles in skin of sheep with black versus white coat color. Immunofluorescence was used to observe the expression localization of differential protein. Western blot and quantitative real time polymerase chain reaction (qRT-PCR) were used to evaluate their role in the coat color formation of sheep. A total of 136 differential proteins were obtained in different coat colors, including 101 up-regulated and 35 down-regulated. Pigmentation function entries were enriched through gene ontology annotation. Tyrosine metabolism and platelet activation signaling pathway were extracted by Kyoto encyclopedia of genes and genomes analysis. Apolipoprotein A-1 (APOA1) and fibrinogen alpha chain (FGA) were found to be critical differential proteins by the interaction of differential proteins in the direct-interaction network diagram. Strikingly, twenty candidate differential proteins were screened, from which beta-actin (ACTB) protein showed higher expression in white sheep skin, while albumin (ALB), APOA1, MAOA (amine oxidase) and FGA proteins showed higher expression in black sheep skin, which was validated by immunofluorescence, western blot, and qRT-PCR. This study identified several novel proteins that may be involved in the coat color formation of sheep. The white and black sheep skin proteome profiles obtained provide a valuable resource for future research to understand the network of protein expression controlling skin physiology and melanogenesis in sheep.

Differentially expressed proteins and microbial communities of the skin regulate disease resistance to Chinese tongue sole (Cynoglossus semilaevis).

Vibriosis, caused by Vibrio, seriously affects the health of fish, shellfish, and shrimps, causing large economic losses. Teleosts are represent the first bony vertebrates with both innate and adaptive immune responses against pathogens. Aquatic animals encounter hydraulic pressure and more pathogens, compared to terrestrial animals. The skin is the first line of defense in fish, constituting the skin-associated lymphoid tissue (SALT), which belongs to the main mucosa-associated lymphoid tissues (MALT). However, little is known about the function of immunity related proteins in fish. Therefore, this study used iTRAQ (isobaric tags for relative and absolute quantitation) to compare the skin proteome between the resistant and susceptible families of Cynoglossus semilaevis. The protein integrin beta-2, the alpha-enolase isoform X1, subunit B of V-type proton ATPase, eukaryotic translation initiation factor 6, and ubiquitin-like protein ISG15, were highly expressed in the resistant family. The 16S sequencing of the skin tissues of the resistant and susceptible families showed significant differences in the microbial communities of the two families. The protein-microbial interaction identified ten proteins associated with skin microbes, including immunoglobulin heavy chain gene (IGH), B-cell lymphoma/leukemia 10 (BCL10) and pre-B-cell leukemia transcription factor 1 isoform X2 (PBX2). This study highlights the interaction between skin proteins and the microbial compositions of C. semilaevis and provides new insights into understanding aquaculture breeding research.

Proteome Analysis of Alpine Merino Sheep Skin Reveals New Insights into the Mechanisms Involved in Regulating Wool Fiber Diameter.

Wool fiber is a textile material that is highly valued based on its diameter, which is crucial in determining its economic value. To analyze the molecular mechanisms regulating wool fiber diameter, we used a Data-independent acquisition-based quantitative proteomics approach to analyze the skin proteome of Alpine Merino sheep with four fiber diameter ranges. From three contrasts of defined groups, we identified 275, 229, and 190 differentially expressed proteins (DEPs). Further analysis using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways revealed that pathways associated with cyclic adenosine monophosphate and peroxisome proliferator-activated receptor signaling are relevant to wool fiber diameter. Using the K-means method, we investigated the DEP expression patterns across wool diameter ranges. Using weighted gene co-expression network analysis, we identified seven key proteins (CIDEA, CRYM, MLX, TPST2, GPD1, GOPC, and CAMK2G) that may be involved in regulating wool fiber diameter. Our findings provide a theoretical foundation for identifying DEPs and pathways associated with wool fiber diameter in Alpine Merino sheep to enable a better understanding of the molecular mechanisms underlying the genetic regulation of wool fiber quality.

040 Vildagliptin administration alters mouse skin proteome

Bullous pemphigoid (BP) is a subcutaneous blistering skin disease in which IgG autoantibodies are formed against the hemidesmosomal protein BP180. Epidemiological studies have revealed a strong association between an increased risk of BP and the use of dipeptidyl peptidase 4 inhibitors (DPP4i/gliptins), especially vildagliptin. We performed a proteomic study on vildagliptin-exposed mice to reveal factors for the development of DPP4i-associated BP. We administered vildagliptin via drinking water to mice (control group, n = 5, treatment group n = 6) for three months and used minimum two-dimensional difference gel electrophoresis (2D-DIGE) to study the effects of vildagliptin on the skin proteome.

Potato tuber skin proteome is enriched with defensive components to prevent the further infection of common scab into tuber flesh

Potato common scab is a soil-borne disease caused mainly by Streptomyces scabies that seriously affects tuber quality and production. To understand the potential molecular mechanisms of potato tuber responded to common scab, a comparative proteomics approach was applied to analyze the proteome alteration of tuber skin and flesh after infected with common scab by two-dimensional gel electrophoresis (2-DE). Quantitative image analysis showed that the abundance of 36 and 26 protein spots in tuber skin and flesh of scab-infected potato were significantly altered (p < 0.05) more than 1.5-fold, respectively. All these differentially abundant proteins were successfully identified by MALDI-TOF/TOF MS, which were mainly involved in defense, bioenergy and metabolism, storage, chaperones, redox homeostasis, miscellaneous and other unknown functions. Compared with tuber flesh, more defense-related proteins were up-regulated in tuber skin, and several cell wall reorganization and lignification-related enzymes were also up-regulated in tuber skin. It appeared that the tuber skin as a barrier might be enriched with components of defense response to prevent the further infection of pathogen into tuber flesh. Several chaperones were up-regulated in tuber skin and flesh, suggesting a key role in reconstructing normal protein conformation and cell homeostasis in tubers. Conversely, common scab causes the down-regulation of many patatin proteins, which might inhibit tuber swelling. This study would provide some new insight into the potential response mechanisms of tuber infected with common scab, and help in developing strategies to improve resistance to common scab in potato breeding.

Spatially resolved proteomic map shows that extracellular matrix regulates epidermal growth

Human skin comprises stratified squamous epithelium and dermis with various stromal cells and the extracellular matrix (ECM). The basement membrane (BM), a thin layer at the top of the dermis, serves as a unique niche for determining the fate of epidermal stem cells (EpSCs) by transmitting physical and biochemical signals to establish epidermal cell polarity and maintain the hierarchical structure and function of skin tissue. However, how stem cell niches maintain tissue homeostasis and control wound healing by regulating the behavior of EpSCs is still not completely understood. In this study, a hierarchical skin proteome map is constructed using spatial quantitative proteomics combined with decellularization, laser capture microdissection, and mass spectrometry. The specific functions of different structures of normal native skin tissues or tissues with a dermatologic disease are analyzed in situ. Transforming growth factor-beta (TGFβ)-induced protein ig-h3 (TGFBI), an ECM glycoprotein, in the BM is identified that could enhance the growth and function of EpSCs and promote wound healing. Our results provide insights into the way in which ECM proteins facilitate the growth and function of EpSCs as part of an important niche. The results may benefit the clinical treatment of skin ulcers or diseases with refractory lesions that involve epidermal cell dysfunction and re-epithelialization block in the future.

Changes in the Skin Proteome and Signs of Allostatic Overload Type 2, Chronic Stress, in Response to Repeated Overcrowding of Lumpfish (Cyclopterus lumpus L.)

Lumpfish (Cyclopterus lumpus L) is a North Atlantic species harvested for its roe and increasingly used as a cleanerfish in Atlantic salmon (Salmo salar L.) farming to remove salmon louse (Lepeophtheirus salmonis). In aquaculture, the health and welfare of fish depends on optimal levels of several biotic and abiotic factors. Crowding, a common abiotic stress factor in aquaculture practice, can affect the welfare and survival of fish. In this study, lumpfish was exposed to crowding stress daily at random timepoints for one month (stress group) or no crowding (control group). Blood and skin were sampled weekly for physiological parameter analysis and proteomics, respectively. Adrenocorticotropic hormone (ACTH) stimulation and dexamethasone (DEX) suppression test were conducted at the sampling timepoints. Gel-based proteomics coupled with liquid chromatography and tandem mass spectrometry (LC-MS/MS) was used to identify protein changes in skin tissues of lumpfish under crowding. The results indicated that the stress group showed signs of allostatic overload type 2 (chronic stress) due to oversensitivity to ACTH, and a reduced negative feedback system with increased baseline levels of cortisol. These chronic changes in the endocrine system promoted changes in secondary and tertiary stress responses as reduced osmoregulatory capacity and stunted growth, after 14 days of stress and onward. Calmodulin, guanine nucleotide binding protein subunit beta 2, glutathione-S-transferase Mu 3, fatty acid binding protein, heat shock cognate 70 kDa protein, keratin, histone H4 and 14-3-3 alpha/beta showed protein spot intensity changes compared with controls in lumpfish skin at one or several time points during the one month period of crowding stress. The differentially expressed proteins are related to several metabolic pathways and are involved in stress and immune responses. Overall, the study shows that lumpfish can suffer from chronic stress with possible dire consequences for the animal welfare.

Oxidative Stress Modulation by Carnosine in Scaffold Free Human Dermis Spheroids Model: A Proteomic Study.

Carnosine is an endogenous β-alanyl-L-histidine dipeptide endowed with antioxidant and carbonyl scavenger properties, which is able to significantly prevent the visible signs of aging and photoaging. To investigate the mechanism of action of carnosine on human skin proteome, a 3D scaffold-free spheroid model of primary dermal fibroblasts from a 50-year-old donor was adopted in combination with quantitative proteomics for the first time. The label free proteomics approach based on high-resolution mass spectrometry, integrated with network analyses, provided a highly sensitive and selective method to describe the human dermis spheroid model during long-term culture and upon carnosine treatment. Overall, 2171 quantified proteins allowed the in-depth characterization of the 3D dermis phenotype during growth and differentiation, at 14 versus 7 days of culture. A total of 485 proteins were differentially regulated by carnosine at 7 days, an intermediate time of culture. Of the several modulated pathways, most are involved in mitochondrial functionality, such as oxidative phosphorylation, TCA cycle, extracellular matrix reorganization and apoptosis. In long-term culture, functional modules related to oxidative stress were upregulated, inducing the aging process of dermis spheroids, while carnosine treatment prevented this by the downregulation of the same functional modules. The application of quantitative proteomics, coupled to advanced and relevant in vitro scaffold free spheroids, represents a new concrete application for personalized therapies and a novel care approach.

150 Skin proteom protection through the chaperone-like activity of an A. agilis extract

Proteom encompasses the full set of proteins in a cell and in an organism. It is a highly dynamic system that maintains its homeostasis, called proteostasis. In case of loss of proteostasis, impaired proteins accumulate, aggregate and/or saturate detoxifying systems leading to proteotoxicity which is a hallmark of ageing, involved in most age-related diseases and in skin ageing. Thereby, protection of the skin proteome is a key challenge to keep a healthy skin and fight accelerated aging. We discovered and characterized a snow bacteria extract (SBE) that displays protective effect on the proteome under stressful conditions.

The inflammatory proteome of hidradenitis suppurativa skin is more expansive than that of psoriasis vulgaris

Although hidradenitis suppurativa (HS) shares some transcriptomic and cellular infiltrate features with psoriasis, their skin proteome remains unknown. To define and compare inflammatory protein biomarkers of HS and psoriasis skin. We assessed 92 inflammatory biomarkers in HS (n=13), psoriasis (n=11), and control skin (n=11) using Olink high-throughput proteomics. We also correlated HS skin and blood biomarkers using proteomics and RNA sequencing. We identified 57 differentially expressed proteins (DEPs) in lesional psoriasis and 64 DEPs in lesional HS skin, compared to healthy controls. Both HS and psoriasis lesional skin demonstrated a significant upregulation of T helper 1 and T helper 17 proteins. Healthy-appearing perilesional HS skin had 63 DEPs compared to healthy controls. Nonlesional HS and psoriasis skin had 24 and 7 DEPs, respectively, compared to healthy controls. Tumor necrosis factor and 8 other proteins were significantly correlated with clinical severity in perilesional HS skin (2cm from a nodule). Inclusion of only moderate-to-severe patients and the cohort size. HS has a greater inflammatory profile and is more diffusely distributed compared with psoriasis. Proteins correlated with disease severity are potential disease mediators. Perilesional skin is comparably inflamed to lesional skin, suggesting the need to treat beyond skin nodules.

Predicting and characterising protein damage in the extracellular matrix

Chronic UVR exposure of human skin can result in photo-ageing which manifests both externally and internally (as remodelling of skin layers including the extracellular matrix-rich dermis). However, the intermittent nature of UVR exposure over a timescale of decades combined with the longevity of many structural dermal proteins makes the identification of dermal photo-ageing targets and mechanisms challenging. Over the past ten years work in our group has demonstrated that: (i) proteins which are rich in amino acid chromophores are susceptible to physiologically relevant doses of solar simulated radiation, (ii) this protein degradation is mediated primarily by the photodynamic production of reaction oxygen species and (iii) UVR-chromophore rich proteins are located in tissue regions where they may act as endogenous sunscreens. We have also shown that ECM proteases selectively degrade UVR-damaged assemblies in vitro and have developed new machine learning-based models to predict protease cleavage sites and hence relative protease susceptibilities in the human skin proteome. The recent development of peptide location fingerprinting applied to conventional mass spectrometry datasets has allowed the identification of novel candidate biomarkers of UVR-induced damage as a consequence of photo-ageing. For example, this approach is able to identify structure-associated modifications in collagen VI alpha chains, although collagen VI remodeling is not evident by conventional histological analysis. These methods and resources (skin proteome database, protein susceptibility prediction and peptide location fingerprinting analysis) are available for use at www.manchesterproteome.manchester.ac.uk. Key areas for future research include using peptide location fingerprinting to: (i) characterise the structural hallmarks of ageing and photo-ageing mechanisms across different phototypes and (ii) evaluate the efficacy of rejuvenating treatments against novel protein biomarkers.

Study of Carnosine’s effect on nude mice skin to prevent UV-A damage

The skin is an important barrier against external attacks from bacteria, radicals, or radiations. UV-A radiations cause significant impairment of this barrier, inducing inflammation, oxidative stress, and wrinkle formation, thereby promoting photoaging. Previous studies reported that carnosine, a potent antioxidant, and carbonyl scavenger agent, may prevent photoaging features in the skin of hairless mice exposed to UV-A radiations. In the present study, we used a quantitative proteomic approach to analyze the changes evoked by carnosine in the skin proteome of hairless mice exposed to UV-A. This approach allowed to quantify more than 2480 proteins, among them consistent differences were observed for 89 proteins in UV-A exposed vs control unexposed skins, and 252 proteins in UV-A-exposed skin preventively treated by carnosine (UVAC) vs UV-A. Several functional pathways were altered in the skins of UV-A exposed hairless mice, including the integrin-linked kinase, calcium signaling, fibrogenesis, cell migration and filament formation. An impairment of mitochondrial function and metabolism was observed, with an up-regulation of cytochrome C oxidase 6B1 and NADH: ubiquinone oxidoreductase S8. Skins pre-treated by carnosine were prevented from UV-A induced proteome alterations. In conclusion, our study emphasizes the potency of a proteomic approach to identify the consequences of UV radiations in the skins, and points out the capacity of carnosine to prevent the alterations of skin proteome evoked by UV-A.

Proteomic signatures for perioperative oxygen delivery in skin after major elective surgery: mechanistic sub-study of a randomised controlled trial

BackgroundMaintaining adequate oxygen delivery (DO2) after major surgery is associated with minimising organ dysfunction. Skin is particularly vulnerable to reduced DO2. We tested the hypothesis that reduced perioperative DO2 fuels inflammation in metabolically compromised skin after major surgery. MethodsParticipants undergoing elective oesophagectomy were randomised immediately after surgery to standard of care or haemodynamic therapy to achieve their individualised preoperative DO2. Abdominal punch skin biopsies were snap-frozen before and 48 h after surgery. On-line two-dimensional liquid chromatography and ultra-high-definition label-free mass spectrometry was used to characterise the skin proteome. The primary outcome was proteomic changes compared between normal (≥preoperative value before induction of anaesthesia) and low DO2 (<preoperative value before induction of anaesthesia) after surgery. Secondary outcomes were functional enrichment analysis of up/down-regulated proteins (Ingenuity pathway analysis; STRING Protein-Protein Interaction Networks). Immunohistochemistry and immunoblotting confirmed selected proteomic findings in skin biopsies obtained from patients after hepatic resection. ResultsPaired punch skin biopsies were obtained from 35 participants (mean age: 68 yr; 31% female), of whom 17 underwent oesophagectomy. There were 14/2096 proteins associated with normal (n=10) vs low (n=7) DO2 after oesophagectomy. Failure to maintain preoperative DO2 was associated with upregulation of proteins counteracting oxidative stress. Normal DO2 after surgery was associated with pathways involving leucocyte recruitment and upregulation of an antimicrobial peptidoglycan recognition protein. Immunohistochemistry (n=6 patients) and immunoblots after liver resection (n=12 patients) supported the proteomic findings. ConclusionsProteomic profiles in serial skin biopsies identified organ-protective mechanisms associated with normal DO2 after major surgery. Clinical trial registrationISRCTN76894700.

Peptide location fingerprinting reveals modification-associated biomarker candidates of ageing in human tissue proteomes.

Although dysfunctional protein homeostasis (proteostasis) is a key factor in many age‐related diseases, the untargeted identification of structurally modified proteins remains challenging. Peptide location fingerprinting is a proteomic analysis technique capable of identifying structural modification‐associated differences in mass spectrometry (MS) data sets of complex biological samples. A new webtool (Manchester Peptide Location Fingerprinter), applied to photoaged and intrinsically aged skin proteomes, can relatively quantify peptides and map statistically significant differences to regions within protein structures. New photoageing biomarker candidates were identified in multiple pathways including extracellular matrix organisation (collagens and proteoglycans), protein synthesis and folding (ribosomal proteins and TRiC complex subunits), cornification (keratins) and hemidesmosome assembly (plectin and integrin α6β4). Crucially, peptide location fingerprinting uniquely identified 120 protein biomarker candidates in the dermis and 71 in the epidermis which were modified as a consequence of photoageing but did not differ significantly in relative abundance (measured by MS1 ion intensity). By applying peptide location fingerprinting to published MS data sets, (identifying biomarker candidates including collagen V and versican in ageing tendon) we demonstrate the potential of the MPLF webtool for biomarker discovery.

Predicting Proteolysis in Complex Proteomes Using Deep Learning.

Both protease- and reactive oxygen species (ROS)-mediated proteolysis are thought to be key effectors of tissue remodeling. We have previously shown that comparison of amino acid composition can predict the differential susceptibilities of proteins to photo-oxidation. However, predicting protein susceptibility to endogenous proteases remains challenging. Here, we aim to develop bioinformatics tools to (i) predict cleavage site locations (and hence putative protein susceptibilities) and (ii) compare the predicted vulnerabilities of skin proteins to protease- and ROS-mediated proteolysis. The first goal of this study was to experimentally evaluate the ability of existing protease cleavage site prediction models (PROSPER and DeepCleave) to identify experimentally determined MMP9 cleavage sites in two purified proteins and in a complex human dermal fibroblast-derived extracellular matrix (ECM) proteome. We subsequently developed deep bidirectional recurrent neural network (BRNN) models to predict cleavage sites for 14 tissue proteases. The predictions of the new models were tested against experimental datasets and combined with amino acid composition analysis (to predict ultraviolet radiation (UVR)/ROS susceptibility) in a new web app: the Manchester proteome susceptibility calculator (MPSC). The BRNN models performed better in predicting cleavage sites in native dermal ECM proteins than existing models (DeepCleave and PROSPER), and application of MPSC to the skin proteome suggests that: compared with the elastic fiber network, fibrillar collagens may be susceptible primarily to protease-mediated proteolysis. We also identify additional putative targets of oxidative damage (dermatopontin, fibulins and defensins) and protease action (laminins and nidogen). MPSC has the potential to identify potential targets of proteolysis in disparate tissues and disease states.

Evaluation and improvement of protein extraction methods for analysis of skin proteome by noninvasive tape stripping

Analysis of the human skin proteome is key to understand molecular mechanisms maintaining health or leading to diseases of this important organ. For minimal invasive sampling of skin proteomes, the use of self-adhesive tape strips has been successfully applied. However, the methods previously presented were evaluated on different types of skin samples (e.g. healthy, diseased) and used a variety of cell lysis/protein extraction methods, which renders a systematic comparison and thus the identification of the most efficient protocols difficult.Here, we present a study comparing five different approaches for cell lysis and protein extraction from single tape strip biopsies. Extraction using a detergent mix or 1% SDS proved to be most efficient. Further, we replaced protein precipitation by single-pot, solid-phase-enhanced sample preparation (SP3), which strongly enhanced the number of identified proteins. This fully LC-MS compatible methodology provides a fast and reproducible approach for minimal invasive sampling of human skin proteomes. Biological significanceFast and reproducible minimal invasive sampling of human skin proteomes is a major prerequisite for clinical proteomics studies aiming to decipher molecular mechanisms involved in the homeostasis as well as in the development of diseases. By optimization of tape strip sampling, e.g. the introduction of SP3 sample cleanup prior to LC-MS analysis, the presented protocol leads to yet not reported numbers of protein identifications from healthy human skin. Further, due to its efficiency it allows analysis from minimal sample amounts, e.g. from single tape strips, while established protocols relied on pooling of multiple tape strips. This provides the opportunity to perform spatially (lateral) resolved proteome analyses from different depths of the skin by analysis of consecutive strips.

Characterization of the Jumbo Squid (Dosidicus gigas) Skin By-Product by Shotgun Proteomics and Protein-Based Bioinformatics.

Jumbo squid (Dosidicus gigas) is one of the largest cephalopods, and represents an important economic fishery in several regions of the Pacific Ocean, from southern California in the United States to southern Chile. Large and considerable discards of this species, such as skin, have been reported to constitute an important source of potential by-products. In this paper, a shotgun proteomics approach was applied for the first time to the characterization of the jumbo squid (Dosidicus gigas) skin proteome. A total of 1004 different peptides belonging to 219 different proteins were identified. The final proteome compilation was investigated by integrated in-silico studies, including gene ontology (GO) term enrichment, pathways, and networks studies. Potential new valuable bioactive peptides such as antimicrobial, bioactive collagen peptides, antihypertensive and antitumoral peptides were predicted to be present in the jumbo squid skin proteome. The integration of the global proteomics results and the bioinformatics analysis of the jumbo squid skin proteome show a comprehensive knowledge of this fishery discard and provide potential bioactive peptides of this marine by-product.