Enrichment of microsomes from Chinese hamster ovary cells by subcellular fractionation for its use in proteomic analysis

Abstract

Chinese hamster ovary cells have been the workhorse for the production of recombinant proteins in mammalian cells. Since biochemical, cellular and omics studies are usually affected by the lack of suitable fractionation procedures to isolate compartments from these cells, differential and isopycnic centrifugation based techniques were characterized and developed specially for them. Enriched fractions in intact nuclei, mitochondria, peroxisomes, cis-Golgi, trans-Golgi and endoplasmic reticulum (ER) were obtained in differential centrifugation steps and subsequently separated in discontinuous sucrose gradients. Nuclei, mitochondria, cis-Golgi, peroxisomes and smooth ER fractions were obtained as defined bands in 30-60% gradients. Despite the low percentage represented by the microsomes of the total cell homogenate (1.7%), their separation in a novel sucrose gradient (10-60%) showed enough resolution and efficiency to quantitatively separate their components into enriched fractions in trans-Golgi, cis-Golgi and ER. The identity of these organelles belonging to the classical secretion pathway that came from 10-60% gradients was confirmed by proteomics. Data are available via ProteomeXchange with identifier PXD019778. Components from ER and plasma membrane were the most frequent contaminants in almost all obtained fractions. The improved sucrose gradient for microsomal samples proved being successful in obtaining enriched fractions of low abundance organelles, such as Golgi apparatus and ER components, for biochemical and molecular studies, and suitable for proteomic research, which makes it a useful tool for future studies of this and other mammalian cell lines.

Compartmentalized Proteomic Profiling Outlines the Crucial Role of the Classical Secretory Pathway during Recombinant Protein Production in Chinese Hamster Ovary Cells

“…This subcellular proteomics strategy led to the identification of 493 DEPs, of which around 80% have not been described as relevant for protein production, providing hundreds of new targets that can be modified in CHO cells. This high number of new targets reflects the potential of subcellular proteomics over classical approaches to increase proteome coverage and the identification of low abundance cellular proteins. ,, Actually, the 59% proteome coverage of this study exceeds that achieved in other differential proteomic studies (5–31%) ,− which reinforces the importance of subcellular fractionation prior to proteomics to allow enrichment of low abundance of proteins, such as those from the CSP and mitochondria. , The fact that one-third of all DEPs were assigned to the CSP (Figure ) supports the use of this technique and highlights the large number of molecular processes from the secretory pathway that can be considered as a limiting factor for RP production in these cells. It is noteworthy that a quarter of all DEPs came from the microsomal gradient (Figure ), demonstrating the potential of this novel sucrose gradient to study the CSP of mammalian cells …”

“…30,31,33 Actually, the 59% proteome coverage of this study exceeds that achieved in other differential proteomic studies (5−31%) 18,20−22 which reinforces the importance of subcellular fractionation prior to proteomics to allow enrichment of low abundance of proteins, such as those from the CSP and mitochondria. 30,51 The fact that one-third of all DEPs were assigned to the CSP (Figure 4) supports the use of this technique and highlights the large number of molecular processes from the secretory pathway that can be considered as a limiting factor for RP production in these cells. It is noteworthy that a quarter of all DEPs came from the microsomal gradient (Figure 3), demonstrating the potential of this novel sucrose gradient to study the CSP of mammalian cells.…”

“…Besides the whole analysis of all DEPs, the contribution of each subcellular compartment (C1–C10, Figure 3 A) to their identification was also examined ( Figure 3 B). Most DEPs came from compartments C3–C5 and C7, which are mainly enriched in Golgi Apparatus, nuclei, mitochondria, peroxisomes, and ER, 51 showing an over-representation of proteins from the CSP. Upregulated proteins were positioned principally in an unidentifiable compartment (C4) and trans -Golgi, while downregulated ones were highly enriched in cis -Golgi.…”

“…In order to capture the cellular processes from the CSP that could be limiting for protein production, a subcellular fractionation protocol was applied to cells collected during the exponential growth phase, 51 from which 10 subcellular compartments were isolated and their proteins submitted to shot-gun proteomics. This subcellular proteomics strategy led to the identification of 493 DEPs, of which around 80% have not been described as relevant for protein production, providing hundreds of new targets that can be modified in CHO cells.…”

“…It is noteworthy that a quarter of all DEPs came from the microsomal gradient ( Figure 3 ), demonstrating the potential of this novel sucrose gradient to study the CSP of mammalian cells. 51 …”