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Comprehensive proteomic characterization identifies potential molecular targets for therapy and imaging in the Cluster 1 human pheochromocytoma and paraganglioma

ABSTRACT: In order to identify new proteins with a potential to become targets for therapy or tumor imaging in pheochromocytoma and paraganglioma, we performed detailed proteomic analysis of 22 well-characterized human PPGL samples and normal chromaffin tissue from adrenal medulla. A standard quantitative proteomic analysis of tumor tissue was accompanied by specific membrane proteome-aimed methods, namely glycopeptide capture using lectin-affinity (Zielinska 2010) or hydrazide chemistry (Tian 2007) and enrichment of membrane-embedded hydrophobic transmembrane segment (Vit et al 2016 ). The parallel application of these complementary methods recently presented as “Pitchfork strategy”, enables deep quantitative profiling of human membrane proteomes without sacrificing the information on soluble non-membrane proteins (Vit et al 2019).

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Adrenal Medulla

DISEASE(S): Pheochromocytoma,Paraganglioma

SUBMITTER: Ondrej Vit

LAB HEAD: Jiří Petrák

PROVIDER: PXD039788 | Pride | 2023-09-25

REPOSITORIES: Pride

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Project description:Ammonium is a waste product that inhibits cell growth, recombinant protein production, and protein glycosylation in mammalian cell culture. Recent studies have demonstrated that ammonium adversely affects glycosylation-related gene expression in Chinese hamster ovary (CHO) cells. However, since the CHO cell line species has not been fully sequenced, a glycosylation transcriptome analysis is not possible in this cell line. Therefore, to further understand the effects of ammonium on glycosylation-related gene expression, NS0 cells, a mouse myeloma cell line, were cultured under elevated ammonium. NS0 cells are similar to CHO cells, in that the NS0 cells are anchorage-independent and commonly used to commercially produce recombinant proteins. Additionally, DNA microarrays containing all known mouse glycosylation-related genes were available to be used to examine gene expression. NS0 cells were cultured under normal (control), elevated ammonium, elevated salt, and elevated ammonium with proline. It was observed that the control and treatments culture growth rates were not significantly different; however, the final cell densities were significantly different. The DNA microarray data was analyzed using a Welch ANOVA test with a Benjamini and Hochberg false discovery rate correction for the multiple comparisons of the glycosylation-genes. No significant difference in gene expression levels between the four conditions examined were observed. The results of this study demonstrated that NS0 cells, at the gene expression level, are insensitive to ammonium. Thus, the decreased glycosylation observed in NS0 cell cultures at elevated ammonium is likely due to changes in synthesis and degradation enzyme activity. In contrast, CHO cells have decreased glycosylation levels due to decreased sialytransferase gene expression and not increased degradation enzyme activity. Therefore, even though NS0 and CHO cells are both commonly used recombinant hosts for glycoprotein synthesis, it appears that NS0 and CHO cells had different control mechanisms respect to glycosylation-related gene expression under elevated ammonium. NS0 cells (ECACC#85110503), originally from the European Collection of Cell Culture, were donated to Clemson University by Merck, Inc. NS0 cells are a mouse myeloma cell line with lymphoblast morphology, non-secreting clone, and cholesterol auxotroph. NS0 cells cultured under four conditions were examined: Control (C), Ammonium-Stressed (A), Salt-Stressed (S), and Ammonium-Stressed with Proline added to mitigate the negative effects of ammonium (P). Triplicates of each condition were used. The cultures were be monitored during the normal batch growth phase. To identify genes sensitive to ammonium in growing cultures, the 90-h time point was selected for RNA isolation and gene expression analysis. Other culture parameters that were monitored include: Cell density, viability, and glucose.

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Comprehensive proteomic characterization identifies potential molecular targets for therapy and imaging in the Cluster 1 human pheochromocytoma and paraganglioma

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