Dataset Information

Non?invasive proteome?wide quantification of skin barrier?related proteins using label?free LC?MS/MS analysis.

ABSTRACT: A number of epidermal proteins are closely related to skin barrier function, the abnormalities of which can lead to specific skin diseases. These proteins must be quantified to further investigate the changes in the skin barrier between healthy and disease states. However, the non?invasive and proteome?wide quantification of skin proteins without any labelling steps remains a challenge. In this study, 3M medical adhesive tapes were used to obtain skin samples from volunteers. Proteins were extracted from fresh skin samples and digested with trypsin. Each tryptic peptide was analysed in three replicates using liquid chromatography with tandem mass spectrometry analysis and label?free quantification. The data were searched against the Human Universal Protein Resource (UniProt) to match with known proteins. Using this method, 1,157 skin proteins recorded in the UniProt were quantified. A total of 50 identical proteins were identified in the three replicate analyses of all samples with no significant differences in abundance. The results provided an objective metric for further study of skin ageing and various skin diseases. Specifically, the non?invasive proteome?wide method used in this study can be applied to future studies of skin diseases related to barrier destruction by monitoring the changes in the levels of epidermal proteins.

SUBMITTER: Liu M

PROVIDER: S-EPMC7115193 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Non‑invasive proteome‑wide quantification of skin barrier‑related proteins using label‑free LC‑MS/MS analysis.

Liu Mengting M Zhang Jing J Wang Yaochi Y Xin Cong C Ma Jie J Xu Shuangjun S Wang Xiaomeng X Gao Jinping J Zhang Xuejun X Yang Sen S

Molecular medicine reports 20200311 5

A number of epidermal proteins are closely related to skin barrier function, the abnormalities of which can lead to specific skin diseases. These proteins must be quantified to further investigate the changes in the skin barrier between healthy and disease states. However, the non‑invasive and proteome‑wide quantification of skin proteins without any labelling steps remains a challenge. In this study, 3M medical adhesive tapes were used to obtain skin samples from volunteers. Proteins were extra ...[more]

PMID: 32186761

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Project description:A widely used method for protein identification couples prefractionation of protein samples by one-dimensional (1D) PAGE with LC/MS/MS. We developed a new label-free quantitative algorithm by combining measurements of spectral counting, ion intensity, and peak area on 1D PAGE-based proteomics. This algorithm has several improvements over other label-free quantitative algorithms: (i) Errors in peak detection are reduced because the retention time is based on each LC/MS/MS run and actual precursor m/z. (ii) Detection sensitivity is increased because protein quantification is based on the combination of peptide count, ion intensity, and peak area. (iii) Peak intensity and peak area are calculated in each LC/MS/MS run for all slices from 1D PAGE for every single identified protein and visualized as a Western blot image. The sensitivity and accuracy of this algorithm were demonstrated by using standard curves (17.4 fmol to 8.7 pmol), complex protein mixtures (30 fmol to 1.16 pmol) of known composition, and spiked protein (34.8 fmol to 17.4 pmol) in complex proteins. We studied the feasibility of this approach using the secretome of angiotensin II (Ang II)-stimulated vascular smooth muscle cells (VSMCs). From the VSMC-conditioned medium, 629 proteins were identified including 212 putative secreted proteins. 26 proteins were differently expressed in control and Ang II-stimulated VSMCs, including 18 proteins not previously reported. Proteins related to cell growth (CYR61, protein NOV, and clusterin) were increased, whereas growth arrest-specific 6 (GAS6) and growth/differentiation factor 6 were decreased by Ang II stimulation. Ang II-stimulated changes of plasminogen activator inhibitor-1, GAS6, cathepsin B, and periostin were validated by Western blot. In conclusion, a novel label-free quantitative analysis of 1D PAGE-LC/MS/MS-based proteomics has been successfully applied to the identification of new potential mediators of Ang II action and may provide an alternative to traditional protein staining methods.

Dataset Information

Non?invasive proteome?wide quantification of skin barrier?related proteins using label?free LC?MS/MS analysis.

Publications

Non‑invasive proteome‑wide quantification of skin barrier‑related proteins using label‑free LC‑MS/MS analysis.

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