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

Main Article Content

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


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 HaCaT 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 HaCaT cells and HaCaT cells exposed to SDS. Using the Metascape database (, an enrichment analysis of GO terms of the Biological Process category of chromosome 18 gene encoded proteins of HaCaT 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 HaCaT 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 HaCaT samples subjected to osmotic shock and cleavage by trypsin in solution.

Article Details

How to Cite
Kisrieva, Y., Samenkova, N., Shkrigunov, T., Larina, O., Rusanov, A., Luzgina, N., Kazieva, L., Karuzina, I., & Petushkova, N. (2023). Comparative Analysis of the Proteomic Profile of HaCaT Keratinocytes Using a 1DE Concentrating Gel. Biomedical Chemistry: Research and Methods, 6(2), e00180.


  1. Ramadan, Q., Ting, F.C. (2016) In vitro micro-physiological immune-competent model of the human skin. Lab. Chip, 16(10), 1899-1908. DOI
  2. OECD (2013), Test No. 431: In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method, OECD Publishing, Paris,34 p. DOI
  3. OECD (2013), Test No. 439: In Vitro Skin Irritation - Reconstructed Human Epidermis Test Method, OECD Publishing, Paris,21 p. DOI
  4. Rusanov, A.L., Luzgina, N.G., Lisitsa, A.V. (2017) Sodium Dodecyl Sulfate Cytotoxicity towards HaCaT Keratinocytes: Comparative Analysis of Methods for Evaluation of Cell Viability. Bulletin of Experimental Biology and Medicine, 163(2), 284-288. DOI
  5. Lindberg, M., Forslind, B., Sagstrom, S., Roomans, G.M. (1992) Elemental changes in guinea pig epidermis at repeated exposure to sodium lauryl sulfate. Acta Dermato-Venereologic, 72(6), 428–431. DOI
  6. Miura, Y., Hisaki, H., Fukushima, B., Nagai,T., Ikeda, T. (1989) Detergent induced changes in serum lipid composition in rats. Lipids, 24(11), 915–918. DOI
  7. Van de Sandt, J.J., Bos, T.A., Rutten, A.A. (1995) Epidermal cell proliferation and terminal differentiation in skin organ culture after topical exposure to sodium dodecyl sulphate. In Vitro Cell. & Dev. Biol. Animal, 31(10), 761–766. DOI
  8. Petushkova, N.A., Rusanov, A.L. , Zgoda, V.G., Pyatnitskiy, M.A., Larina, O.V., Nakhod, K.V., Luzgina, N.G., Lisitsa, A.V. (2017) Proteome of the human hacat keratinocytes: identification of the oxidative stress proteins after sodium dodecyl sulpfate exposure. Molecular Biology, 51(5), 748–758. DOI
  9. Quirino, J.P. (2018) Sodium dodecyl sulfate removal during electrospray ionization using cyclodextrins as simple sample solution additive for improved mass spectrometric detection of peptides. Anal Chim Acta, 16 (1005), 54-60. DOI
  10. Shevchenko, A., Tomas, H., Havlis, J., Olsen, J.V., Mann, M. (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc., 1(6), 2856-60. DOI
  11. Gold Biotechnology (2018) In-gel digestion and extraction of proteins protocol. Retrieved September 9, 2022 from:
  12. Kachuk, C., Stephen, K., Doucette, A. (2015) Comparison of sodium dodecyl sulfate depletion techniques for proteome analysis by mass spectrometry. J. Chromatography A. DOI
  13. Ilavenil, S., Al-Dhabi, N.A., Srigopalram, S., Kim, Y.O., Agastian, P., Baaru, R., Choi, K.C., Arasu, M.V., Park, C.G., Park, K.H. (2016) Removal of SDS from biological protein digests for proteomic analysis by mass spectrometry. Proteome Sci., 14, 11. DOI
  14. Shkrigunov, T., Pogodin, P., Zgoda, V., Larina, O., Kisrieva, Y., Klimenko, M., Latyshkevich, O., Klimenko, P., Lisitsa, A., Petushkova, N. (2022) Protocol for increasing the sensitivity of MS-based protein detection in human chorionic villi. Curr. Issues Mol. Biol., 44 (5), 2069–2088. DOI
  15. UniProt: the Universal Protein Knowledgebase in 2023. The UniProt Consortium. Nucleic Acids Research, 51 (D1), D523–D531. DOI
  16. Kisrieva, Y.S., Samenkova, N.F., Larina, O.B., Zgoda, V.G., Karuzina, I.I., Rusanov, A.L., Luzgina, N.G.,Petushkova, N.A. (2020) Comparative study of the human keratinocytes proteome of the HaCaT line: identification of proteins encoded by genes of 18 chromosomes under the influence of detergents. Biomeditsinskaya Khimiya, 66(6), 469-476. DOI
  17. Walker, J.M. (1994) The bicinchoninic acid (BCA) assay for protein quantitation. Methods Mol. Biol., 32, 5–8. DOI
  18. Chambers, M., Maclean, B., Burke, R. et al. (2012) A cross-platform toolkit for mass spectrometry and proteomics. Nat. Biotechnol., 30, 918–920. DOI
  19. Vaudel, M., Barsnes, H., Berven, F.S., Sickmann, A, Martens, L. (2011) SearchGUI: An open-source graphical user interface for simultaneous OMSSA and X!Tandem searches. Proteomics, 11(5), 996–999. DOI
  20. Vaudel, M., Burkhart, J., Zahedi, R. et al. (2015) PeptideShaker enables reanalysis of MS-derived proteomics data sets. Nat Biotechnol., 33, 22–24. DOI
  21. Florens, L., Carozza, M. J., Swanson, S.K., Fournier, M., Coleman, M.K., Workman, J. L., Washburn, M.P. (2006) Analyzing chromatin remodeling complexes using shotgun proteomics and normalized spectral abundance factors. Methods., 40(4), 303-311. DOI
  22. Ashburner, M., Ball,C.A., Blake, J.A., Botstein, D., Butler, H., Cherry, J.M., Davis, A.P., Dolinski, K., Dwight, S.S., Eppig, J.T., Harris, M.A., Hill, D.P., Issel-Tarver, L., Kasarskis, A., Lewis, S. & Matese, J.C., Richardson, J.E., Ringwald, M., Rubin, G.M., Sherlock, G. (2000) Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat. Genet., 25(1), 25-9. DOI
  23. Mi, H., Thomas, P. (2009) PANTHER pathway: an ontology-based pathway database coupled with data analysis tools. Methods Mol Biol., 563, 123-40. DOI
  24. Kopylov, A.T., Zgoda, V.G., Archakov, A.I. (2009) Label-free quantitative analysis of proteins using mass-spectrometry. Biomeditsinskaya Khimiya, 55(2), 125-39. DOI
  25. Uhlén, M., Fagerberg, L., Hallström, B.M., Lindskog C. , Oksvold, P., Adil Mardinoglu, A., Sivertsson, Å,. Kampf, C., Sjöstedt, E.(2015). Tissue-based map of the human proteome. Science, 347 (6220). DOI
  26. Oldach, M. (2018) Normalized spectral abundance factor (NSAF) for quantitative liquid chromatography mass spectrometry-based proteomics. GitHub. Retrieved September 9, 2022 from: ' target='_blank' > DOI
  27. Eisenberg, E., Levanon, EY. (2013) Human metang genes, revisited. Trends Genet., 29, 569–574. DOI
  28. Hounkpe, B.W., Chenou, F., de Lima, F., De Paula, E.V. (2021) HRT Atlas v1.0 database: redefining human and mouse housekeeping genes and candidate reference transcripts by mining massive RNA-seq datasets. Nucleic Acids Res., 49 (D1), 947-955. DOI
  29. Lane, L., Argoud-Puy, G., Britan, A., Cusin, I., Duek, P.D., Evalet, O., Gateau, A., Gaudet, P., Gleizes, A., Masselot, A., Zwahlen, C., Bairoch, A. (2012) neXtProt: a knowledge platform for human proteins. Nucleic Acids Res., 40 (Database issue), D76-83. DOI
  30. Wu, Q., Feng, Q., Xiong, Y., Xing, L. (2020) RAB31 is targeted by miR-26b and serves a role in the promotion of osteosarcoma. Oncol. Lett., 20(5), 244. DOI
  31. Tanaka, Ki, Kanazawa, I., Richards, J.B., Goltzman, D, Sugimoto, T. (2020) Modulators of Fam210a and Roles of Fam210a in the Function of Myoblasts. Calcified Tissue International, 106, 533–540 DOI
  32. Poulton, C.J., Schot, R., Kia, S.K., Jones, M., Verheijen, F.W., Venselaar, H., Marie-Claire, de Wit Y., de Graaff, E., Bertoli-Avella, A.M., Mancini, G.M.S. (2011) Microcephaly with simplified gyration, epilepsy, and infantile diabetes linked to inappropriate apoptosis of neural progenitors. Am. J. Hum. Genet., 89(2), 265-76. DOI
  33. Hall, P.A., Russell, S.E.H. (2004) The pathobiology of the septin gene family. J. Pathol., 204(4), 489-505. DOI
  34. Dolat, L., Hunyara, J.L., Bowen, J.R., Spiliotis, E.T. (2014) Septins promote stress fiber-mediated maturation of focal adhesions and renal epithelial motility. Journal of Cell Biology, 207(2), 225-35. DOI
  35. Montagna, C., Bejerano-Sagie, M., Zechmeister, J.R. (2015) Mammalian septins in health and disease. Res. Rep. Biochem., 5, 59–73. DOI 10.2147/RRBC.S59060
  36. Farrugia, A.J., Rodrıguez, J., Orgaz, J.L., Lucas, M., Sanz-Moreno, V. & Calvo, F. (2020) CDC42EP5/BORG3 modulates SEPT9 to promote actomyosin function, migration, and invasion. J. Cell Biol., 219 (9), e201912159. DOI
  37. Zhou,Y., Zhou B., Pache, L., Chang, M., Khodabakhshi, A.H., Tanaseichuk, O., Benner, C., Sumit, K., Chanda, S.K. (2019) Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. Nature Communication, 10(1), 1523. DOI
  38. Heath, C.G., Viphakone, N., Wilson, S.A. (2016) The role of TREX in gene expression and disease. Biochem J., 473(19), 2911–35. DOI
  39. Dominguez-Sanchez, M.S., Saez, C., Japon, M.A., Aguilera, A., Luna, R. (2011) Differential expression of THOC1 and ALY mRNP biogenesis/export factors in human cancers. BMC Cancer, 11(77). DOI
  40. Huber, O., Petersen, I. (2015) 150th anniversary series: desmosomes and the hallmarks of cancer. Cell Commun Adhes., 22(1), 15–28. DOI
  41. Takeda, A., Kajiya, A., Iwasawa, A., Nakamura, Y., Hibino, T. (2002) Aberrant expression of serpin squamous cell carcinoma antigen 2 in human tumor tissues and cell lines: evidence of protection from tumor necrosis factor mediated apoptosis. Biol. Chem., 383, 1231–1236. DOI
  42. Tonnetti, L., Netzel-Arnett, S., Darnell, G.A., Hayes, T., Buzza, M.S., Anglin, I.E., Suhrbier, A., Antalis, T.M. (2008) SerpinB2 protection of retinoblastoma protein from calpain enhances tumor cell survival. Cancer Res., 68, 5648–5657. DOI
  43. Ding, S., Blue, R.E., Morgan, D.R., Lund, P.K. (2014) Comparison of multiple enzyme activatable near-infrared fluorescent molecular probes for detection and quantification of inflammation in murine colitis models. Inflamm. Bowel Dis., 20(2), 363-77. DOI
  44. Askew, Y.S., Pak, S.C., Luke, C.J., Askew, D.J., Cataltepe, S., Mills, D.R., Kato, H., Lehoczky, J., Dewar, K., Birren, B., Silverman, G.A. (2001) SERPINB12 is a novel member of the human ov-serpin family that is widely expressed and inhibits trypsin-like serine proteinases. J. Biol. Chem., 276(52), 49320-30. DOI