Biomedical Chemistry: Research and Methods 2023, 6(2), e00180

Comparative Analysis of the Proteomic Profile of HaCaT Keratinocytes Using a 1DE Concentrating Gel

I.S. Kisrieva*, N.F. Samenkova, T.S. Shkrigunov, O.B. Larina, А.L. Rusanov, N.G. Luzgina, L.Sh. Kazieva,
I.I. Karuzina, N.A. Petushkova

Institute of Biomedical Chemistry, 10 Pogodinskaya str., Moscow, 119121 Russia; e-mail: juliaks@bk.ru

Keywords:keratinocytes НаСаТ; SDS extracts; 1DE-gel concentration, in-gel tryptic digestion, SearchGUI; X! Tandem; MS-GF+, proteins encoded by genes of chromosome 18; LC-MS/MS

DOI:10.18097/BMCRM00180

The whole version of this paper is available in Russian.

A sample preparation protocol for proteomic profiling of HaCaT keratinocyte was evaluated. It is based on protein solubilization in the presence of 0.2% SDS, a 1DE-gel concentration procedure (SDS-PAGE without fractionation in the separating gel), and in gel cleavage by trypsin followed by LC-MS/MS. Using tandem mass spectrometry with electrospray ionization, a comparative analysis of HaCaT keratinocyte proteins was carried out before and after exposure of cells to sodium dodecyl sulfate (25 mg/ml) for 48 hours; proteins encoded by human chromosome 18 genes were chosen as the comparison proteins.  A total of 2418 proteins were detected in the НaСaT immortalized human keratinocytes, 70% of these proteins were identified by two or more unique peptides. Panoramic mass spectrometry analysis identified 38 proteins encoded by chromosome 18 genes, 27 proteins were common to control НaСaT cells and НaСaT cells exposed to SDS. Using the Metascape database (https://metascape.org), an enrichment analysis of GO terms of the Biological Process category of chromosome 18 gene encoded proteins of НaСaT keratinocytes was performed before and after the SDS exposure. The SDS exposure resulted in a slight enrichment of the GO term "response to stimulus" (GO:0050896) and the related GO term "negative regulation of biological process" (GO:0048519). We found decreased expression levels of membrane proteins encoded by chromosome 18 genes related to cell-cell adhesion (GO:0098609), such as DSC1, DSC3, and DSG1. A decrease in the expression level of desmosomal cadherins is characteristic of malignant neoplasms developing from epithelial tissue cells of various internal organs, mucous membranes, and skin. The method of preparation of НaСaT keratinocyte samples used in this work increased the sensitivity of proteomic analysis of cell culture and made it possible to identify twice as many proteins in one gel strip as compared to the number of proteins (1284) in НaСaT samples subjected to osmotic shock and cleavage by trypsin in solution.
Figure 1. Mass spectrum and fragment ions of the peptide 281YTCHVQHEGLPEPLTLR charge 3+, 684.01 m/z) which  is specific to the Putative HLA class I histocompatibility antigen, alpha chain H (P01893) missing protein encoded on human chromosome 6, identified using an open-source graphical user interface SearchGUI with simultaneous integrated search algoritms X!Tandem and MS-GF+.
Figure 2. Violin plots of some parameters for identifying proteins encoded by human chromosome 18 genes in HaCaT cells before and after exposure to SDS. (a) Distribution of unique peptides per protein; (b) Distribution of NSAF values. Gray color - control cells, blue color – keratinocytes after exposure to SDS. The black dot indicates the mean value of the parameter, the red dot is the median, and the vertical black line is the standard deviation.
Figure 3. GO enrichment analysis (Biological Process) using Gene Ontology Resource (http://geneontology.org/) and The PANTHER (Protein ANalysis THrough Evolutionary Relationships) (http://pantherdb.org).
Figure 4. Enrichment Analysis: heatmap visualization of proteins encoded by genes of of human chromosome 18 identified in SDS-treated samples of HaCaT human keratinocytes using the Metascape database (http://metascape.org). A heatmap using a discrete color scale illustrating statistical significance is presented for the main enrichment clusters of Gene Ontology (GO) in the biological processes category. Gray color indicates lack of significance.

CLOSE
Table 1. The list of proteins encoded by human chromosome 18 genes and identified in control and SDS-treated samples of HaCaT human keratinocytes.

FUNDING

The work was done in the framework of the Russian Federation fundamental research program for the long-term period for 2021-203 (№122030100168-2). Mass-spectrometry measurements were performed using the equipment of “Human Proteome” Core Facilities of the Institute of Biomedical Chemistry (Russia).

Supplementary materials are available at http://dx.doi.org/10.18097/BMCRM00180

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