Identification of differentially regulated deceitful proteins in SH‐SY5Y cells engineered with Tet‐regulated protein expression system

Abstract

Tetracycline regulated protein expression in mammalian cells is a powerful tool to predict the physiological function, cellular localization, and stability of a protein. In addition, to predict metabolic networks affected by the expression of wild-type or mutant forms of proteins, researchers generally produce a single mammalian cell clone that can express the protein of interest under tetracycline control and study the changes occurring in overall proteome before and after expression of a protein of interest. One limitation of tetracycline regulated clonal cell creation, however, is that it sometimes creates clones with changed protein levels even without the expression of the protein of interest due to the nonspecific insertion of the gene encoding the protein of interest into the genome or disruption of a metabolic pathway due to insertional silencing or activation. The aim of this study was to demonstrate the limitation of tetracycline regulated gene expression by creating clonal cell lines expressing the wild-type or the mutant forms of Fat mass and obesity-associated protein. Comparative proteome analysis of the protein extracts by two-dimensional gel electrophoresis coupled to MALDI-TOF/TOF revealed the presence of eight proteins subjected to differential regulation even in the absence of induction. The identified proteins were 14-3-3 protein Epsilon, Vimentin, Heterogeneous nuclear ribonucleoprotein K, Tubulin beta-2C chain, Heat shock protein HSP 90-alpha, Heat shock protein HSP 90-beta, Alpha-enolase, TATA-binding protein-associated factor 2N. An ultimate care should be taken to prevent reporting of deceitful proteins generated from studies utilizing tetracycline regulated gene expression systems.

Fat Mass and Obesity Associated (FTO) Protein Ekspresyonunun Neden Olduğu SH-SY5Y Hücrelerinin Proteomunda Meydana Gelen Değişiklikler, FTO Proteininin Çok Yönlü Özellikleri Ortaya Çıkarır

“…In most studies using tet-regulated protein expression systems, a single stable clone is used without considering the fact that differences may occur between different clones. 31 Therefore, the results provided here were highly confident in providing a snapshot of the changes that occurred during expressions of the WT and the mutant FTO proteins. The overall evaluation of the data implicated that the soluble proteome of SH-SY5Y cells were not profoundly affected by the expressions of the WT or the mutant FTO proteins.…”

“…The WT-FTO expressing clones were created and labeled as clones #3, #5, #12 for the WT in our previous study. 31 Three of the screened colonies expressing the mutant FTO proteins were propagated and labeled as clones #2, #3, #5 for the mutant.…”

Are Mannan-binding Lectine Serin Protease-2 and Alpha-1-microglobulin and Bukinin Precursor the Potential Biomarkers of Manic Episode? A Study via Urinary Proetomic Analysis

“…In-gel tryptic digestion of the proteins was performed using an in-gel digestion kit (Thermo Fisher Scientific, Waltham, MA, USA) as described in Kanli et al [15]. Protein identification experiments were with ABSCIEX 5800 MALDI-TOF/TOF system (AB Sciex, Framingham, MA, USA).…”

Identification of Novel Proteins from Black Cumin Seed Meals Based on 2D Gel Electrophoresis and MALDI-TOF/TOF-MS Analysis

“…A database was created using the entries in NCBI-NR databank (https://www.ncbi. nlm.nih.gov/refseq/about/nonredundantproteins) and implemented into the MASCOT search engine [17] including the following search criteria: enzyme of trypsin; at least five independent peptides matched; at most one missed cleavage site; MS tolerance set to ±50 ppm and MS/MS tolerance set to ±0.4 Da; fixed modification being carbamidomethylation (Cys) and variable modification being oxidation (Met); peptide charge of 1+ and being monoisotopic. Only significant hits, as defined by the MASCOT probability analysis (푃 < 0.05), were accepted.…”