Dataset Information

Novel skin photoageing biomarkers of epidermis and dermis revealed by traditional relative quantification in combination with a novel peptide locational fingerprinting MPLF.

ABSTRACT: Human skin undergoes profound structural remodelling following chronic exposure to ultraviolet radiation (UVR) – photoageing - that is normally studied by comparing photoexposed and photoprotected skin sites. Whilst it is well established that the abundance and distribution of major dermal extracellular matrix (ECM) proteins (principally collagen I and elastin) are compromised in skin, the influence of UVR exposure on other skin protein constituents, including epidermal proteins, is poorly understood. We used label-free LC-MS/MS proteomics to characterise the proteomic profiles of intrinsically aged buttock and photoaged forearm epidermis and dermis. Besides traditional shotgun proteomics we have also developed and used novel proteomics analysis approach to reveal locational changes in peptide yields from proteins named Manchester Peptide Locational Fingerprinting (MPLF) which is based on spectral counting. MPLF is a new proteomic analysis tool that can identify structural modification-associated differences within proteins from label-free liquid chromatography tandem mass spectrometry (LC-MS/MS) datasets generated from biological samples. We have identified a total of 174 dermal proteins and 146 epidermal proteins with MPLF while relative quantification revealed a total of 635 dermal proteins and 926 epidermal proteins significantly different between photoaged forearm and intrinsically-aged buttock according to peak area intensity.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Skin

SUBMITTER: Alexander Eckersley

LAB HEAD: Michael J Sherratt

PROVIDER: PXD021194 | Pride | 2021-04-09

REPOSITORIES: Pride

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Publications

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

Ozols Matiss M Eckersley Alexander A Mellody Kieran T KT Mallikarjun Venkatesh V Warwood Stacey S O'Cualain Ronan R Knight David D Watson Rachel E B REB Griffiths Christopher E M CEM Swift Joe J Sherratt Michael J MJ

Aging cell 20210408 5

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 ...[more]

PMID: 33830638

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Project description:To obtain a separation of the epidermal and dermal compartments in order to examine compartment specific biological mechanisms in the skin we incubated 4 mm human skin punch biopsies in ammonium thiocyanate (NH4SCN). We wanted to test 1) the histological quality of the dermo-epidermal separation obtained by different incubation times 2) the amount and quality of extractable epidermal RNA, and 3) its impact on sample RNA expression profiles assessed by large-scale gene expression microarray analysis in both normal and inflamed skin. At 30 minutes incubation, the split between dermis and epidermis was not always histologically well-defined (i.e. occurred partly intra-epidermally) but varied between subjects. Consequently, curettage along the dermal surface of the biopsy was added to the procedure. This modified method resulted in an almost perfect separation of the epidermal and dermal compartments and satisfactory amounts of high-quality RNA were obtained. Hybridization to Affymetrix HG_U133A 2.0 GeneChips showed that ammonium thiocyanate incubation had a minute effect on gene expression resulting in only one significantly downregulated gene (cystatin E/M). We conclude that epidermis can be reproducibly and almost completely separated from the dermis of 4 mm skin biopsies by 30 min incubation in 3.8% ammonium thiocyanate combined with curettage of the dermal surface, producing high-quality RNA suitable for transcriptional analysis. Our refined method of dermo-epidermal separation will undoubtedly prove valuable in the many different settings, where the epidermal and dermal compartments need to be evaluated separately. Upper buttock skin in 4 healthy subjects was exposed to sodium lauryl sulphate, or sampled directly. For each subject, 4 biopsies were obtained: Two from inflamed skin, and two from adjacent normal skin. One irritated and one normal skin sample was placed directly in RNAlater. The remaining two samples were incubated in ammonium thiocyanate for 30 minutes at RT and then placed in RNAlater without performing any separation of the dermal and epidermal layers. This was done to investigate the effect of 30 minutes treatment with ammonium thiocyanate on both inflamed and non-inflamed skin. Data was normalized with quantile method (matrix 1) Forearm biopsies from 13 volunteers were separated to epidermis and dermis by use of ammonium thiocyanate. For comparison of full skin and epidermis without irritation, data from identical probe sets from HG_U133A 2.0 and HG_U133 plus 2.0 was extracted and normalised as one data set using quantile method (matrix 2).

Dataset Information

Novel skin photoageing biomarkers of epidermis and dermis revealed by traditional relative quantification in combination with a novel peptide locational fingerprinting MPLF.

Publications

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

Similar Datasets

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