An optimised method for the proteomic profiling of full thickness human skin

Bliss et al. Biological Procedures Online (2016) 18:15 DOI 10.1186/s12575-016-0045-y METHODOLOGY Open Access An optimised method for the proteomic profiling of full thickness human skin Emily Bliss1* , Wendy E. Heywood1, Malika Benatti2, Neil J. Sebire2 and Kevin Mills1 Abstract Background: The skin is the largest organ of the human body and is the first line barrier defence against trauma, microbial infiltration and radiation. Skin diseases can be a result of multi-systemic disease or an isolated condition. Due to its proteolysis resistant properties there are relatively few human skin proteomic datasets published compared with other human organs or body fluids. Skin is a challenging tissue to analyse using traditional proteomic techniques due to its high lipid content, insolubility and extensive cross-linking of proteins. This can complicate the isolation and digestion of proteins for analysis using mass spectrometry techniques. Results: We have optimised a sample preparation procedure to improve solubilisation and mass spectral compatibility of full thickness skin samples. Using this technique, we were able to obtain data for the proteome profile of full thickness human skin using on-line two-dimensional liquid chromatography, followed by ultra-high definition label-free mass spectrometry analysis (UDMSE). We were able to identify in excess of 2000 proteins from a full thickness skin sample. Conclusions: The adoption of on-line fractionation and optimised acquisition protocols utilising ion mobility separation (IMS) technology has significantly increased the scope for protein identifications ten-fold. Keywords: Proteomics, Skin profiling, Mass spectrometry, Solubilisation Background The skin, being the largest organ of our bodies is of paramount importance for barrier protection and first line defence against trauma, microbial infiltration and radiation. However when this barrier is damaged or diseased numerous dermatological conditions can arise. These diseases by their nature are superficial, visible and can look unpleasant, which can contribute to a significant psychological and social burden [1]. To understand more about diseases affecting the skin and the skin barrier in particular, it is important that we understand the complex composition of proteins, metabolites and lipids that make up the skin and how they play a functional role in its overall structure. The skin consists of three major distinct layers; the epidermis, dermis and the subcutaneous fat layer. The purpose of the subcutaneous fat layer is largely for insulation, the dermis is predominantly made up of collagens, elastin * Correspondence: 1 Centre for Translational Omics, UCL Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK Full list of author information is available at the end of the article and fibrillin as well as nerve endings, sweat glands, sebaceous glands, hair follicles and major blood vessels providing nutrients for the epidermal layer [2]. The epidermis is the most superficial layer of the skin and comprises the skin barrier; despite being the thinnest of all three layers it is arguably the most complex [3]. Despite the importance of the skin there are relatively few human skin proteomic studies and datasets currently available in the literature [4] compared with other human organs or body fluids. A human-specific PubMed search shows that there are three times as many publications for “liver AND proteomics” [5] and twelve times as many publications for “blood AND proteomics” [6] compared with publications for “skin AND proteomics” [7]. Skin is a challenging tissue to analyse using traditional proteomic techniques due to its high lipid content, insolubility and extensive cross-linking of proteins. This can complicate the isolation and digestion of proteins for analysis using mass spectrometry techniques. Techniques used by research groups in the current selection of © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bliss et al. Biological Procedures Online (2016) 18:15 available publications about skin proteomics [7] include gel-based protein fractionation [8, 9], heavy isotope labelled assays to identify phosphorylated proteins [10] and studying the proteins secreted by the skin, rather than the skin tissue itself [11]. The latter two techniques are specific to a particular aspect of the skin proteome and do not represent the composition of the skin tissue as a whole. Gel-based protein isolation or fractionation techniques is a viable method for preparing samples prior to mass spectrometry analysis and one which we explored whilst developing the method presented in this paper. When comparing a gelbased fractionation technique and the on-line fractionation technique described here we found a 50 % increase in the number of proteins detected (data not shown). Here we describe an optimised method of fractionating, chromatographically separating and detecting proteins from a trypsin digested sample of human skin. This method is relatively quick, consists of less sample preparation steps and less protein (3 μg) compared with similar gel-based methods. Results Previously in our laboratory we have been able to identify 100–200 proteins from a full thickness skin biopsy [12], however with an upgraded system and modified Page 2 of 7 methods we have improved this protocol and achieved a ten-fold increase in the number of identified proteins. In order to be a fair comparison the results presented in this section are from the same sample of skin tissue and are representative of the number of proteins detected by these methods from other samples of skin (data not shown). In the first instance the skin preparation method comprised of powdering the frozen tissue using a pestle and mortar, further homogenisation using a glass homogeniser, sonication, filtration of interfering low molecular weight compounds. This was a method adapted from other published proteomics methods for other tissues [13–15]. Peptides would then be separated using reverse phase chromatography a nanoAcquity liquid chromatography system coupled to a quadrupole time-of-flight mass spectrometer (QToF Premier, Waters, Manchester) [12] (Fig. 1). Using this workflow 218 proteins were identified from the skin samples as detailed in Additional file 1: Table S1. Modifying the initial preparation method (Fig. 2) improved the number of detected proteins, to 2237 (...truncated)