Supporting information for tryptic EPO glycopeptide LC-MS analysis via cysteine aminoethylation
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap Velos, Q Exactive HF-X hybrid quadrupole-Orbitrap
ORGANISM(S): Cho Cells
SUBMITTER: 
Manfred Wuhrer
PROVIDER: MSV000085318 | MassIVE | Tue Apr 21 14:32:00 BST 2020
REPOSITORIES: MassIVE
Publications
Cysteine Aminoethylation Enables the Site-Specific Glycosylation Analysis of Recombinant Human Erythropoietin using Trypsin.
Lippold Steffen S Büttner Alexander A Choo Matthew S F MSF Hook Michaela M de Jong Coen J CJ Nguyen-Khuong Terry T Haberger Markus M Reusch Dietmar D Wuhrer Manfred M de Haan Noortje N
Analytical chemistry 20200709 14
Recombinant human erythropoietin (rhEPO) is an important biopharmaceutical for which glycosylation is a critical quality attribute. Therefore, robust analytical methods are needed for the in-depth characterization of rhEPO glycosylation. Currently, the protease GluC is widely established for the site-specific glycosylation analysis of rhEPO. However, this enzyme shows disadvantages, such as its specificity and the characteristics of the resulting (glyco)peptides. The use of trypsin, the gold sta ...[more]
Similar Datasets
Project description:Irani2015 - Genome-scale metabolic model of
P.pastoris N-glycosylation
This model is described in the article:
Genome-scale metabolic model
of Pichia pastoris with native and humanized glycosylation of
recombinant proteins.
Irani ZA, Kerkhoven E, Shojaosadati
SA, Nielsen J.
Biotechnol. Bioeng. 2015 Oct;
Abstract:
Pichia pastoris is used for commercial production of human
therapeutic proteins, and genome-scale models of P. pastoris
metabolism have been generated in the past to study the
metabolism and associated protein production by this yeast. A
major challenge with clinical usage of recombinant proteins
produced by P. pastoris is the difference in N-glycosylation of
proteins produced by humans and this yeast. However, through
metabolic engineering a P. pastoris strain capable of producing
humanized N-glycosylated proteins was constructed. The current
genome-scale models of P. pastoris do not address native nor
humanized N-glycosylation, and we therefore developed
ihGlycopastoris, an extension to the iLC915 model with both
native and humanized N-glycosylation for recombinant protein
production, but also an estimation of N-glycosylation of P.
pastoris native proteins. This new model gives a better
predictions of protein yield, demonstrates the effect of the
different types of N-glycosylation of protein yield, and can be
used to predict potential targets for strain improvement. The
model represents a step towards a more complete description of
protein production in P. pastoris, which is required for using
these models to understand and optimize protein production
processes. This article is protected by copyright. All rights
reserved.
This model is hosted on
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MODEL1510220000.
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2024-10-15 | BIOMD0000001096 | BioModels
Project description:These research areas concentrate on stress induced proteases in recombinant Escherichia coli, glycosylation heterogeneity due to bioprocess conditions produced in mammalian cells, and metabolic engineering of E. coli. The hypothesis of this project is that recombinant protein glycosylation is inefficient under normal bioreactor conditions since the additional glycosylation reactions necessary for the recombinant protein exceed the metabolic capacity of the cells. Normal bioreactor conditions have been optimized for cell growth, and sometimes for protein productivity. Only recently has it been accepted that optimal glycosylation may not occur under optimal growth or protein productivity conditions. Specific Aim: Determine the relationship between bioreactor conditions and glycosylation gene expression in NS0 cells.
2011-04-29 | GSE28920 | GEO
Project description:Raw data of plasma-derived total and HLA-A2-specific IgG Fc N-glycosylation analyzed by RP-LC-MS.
| MSV000090430 | MassIVE
Project description:These research areas concentrate on stress induced proteases in recombinant Escherichia coli, glycosylation heterogeneity due to bioprocess conditions produced in mammalian cells, and metabolic engineering of E. coli. The hypothesis of this project is that recombinant protein glycosylation is inefficient under normal bioreactor conditions since the additional glycosylation reactions necessary for the recombinant protein exceed the metabolic capacity of the cells. Normal bioreactor conditions have been optimized for cell growth, and sometimes for protein productivity. Only recently has it been accepted that optimal glycosylation may not occur under optimal growth or protein productivity conditions. Specific Aim: Determine the relationship between bioreactor conditions and glycosylation gene expression in NS0 cells. EXPERIMENT: Mouse NS0 myeloma cells were grown in culture, stressed with 5 mM NaCl, 10 mM proline plus 5 mM ammonia, or 5 mM ammonia, along with an unstressed control group. The growth of the cultures were followed until the late exponential phase (90 hours), at which time two 50 mLs of cells were harvested and RNA extracted. Samples were prepared in triplicate, for a total of 12 samples. The RNA was amplified and labeled by Microarray Core (E) and hybridized to the GLYCOv3 microarrays.
2011-04-29 | E-GEOD-28920 | biostudies-arrayexpress
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.
2009-08-05 | GSE17495 | GEO
Project description:Heterogeneity of protein glycosylation poses great challenge for analysis that is key to un-puzzle systems glycobiology in diseases. Resolving this conundrum requires global enrichment of glycopeptides for identification and quantitation. To this aim, hydrophilic interaction chromatography (HILIC) has been proved to be an effective approach to enrich N-linked glycopeptides (N-glycopeptides). However, its effectiveness to enrich O-linked glycopeptides (O-glycopeptides) and isobaric labelled glycopeptides remains unclear. Here, we studied three different cartridges in HILIC mode. It was found that removal of N-glycosylation prior to enrichment improved identification of O-glycopeptides. It was noted that increased yield and number of glycosylation identification were seen when using cartridges having materials for strong anion exchange (SAX) chromatography. Remarkably, O-glycopeptides were found to be selectively enriched by HILIC with further improved enrichment being seen when Retain AX cartridge being used. Of particular note, isobaric labelled glycopeptides could be readily enriched by SAX cartridges in HILIC mode to enable quantitative glycoproteomics. It is anticipated that the use of SAX cartridges will facilitate broad applications of qualitative and quantitative glycoproteomics in diseases.
2017-10-19 | PXD005145 | Pride
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.
2010-05-15 | E-GEOD-17495 | biostudies-arrayexpress
Project description:In male teleosts, testicular steroids are essential hormones for the regulation of spermatogenesis and their production is regulated by pituitary gonadotropins. In the Senegalese sole (Solea senegalensis), an economically important flatfish with semi-cystic and asynchronous spermatogenesis, the endocrine mechanisms involved in the regulation of spermatogenesis, particularly regarding the production and regulation of testicular steroids, are not well understood.This study aimed at describing the transcriptomic changes taking place in the Senegalese sole testis in response to hCG stimulation in vivo. Gene expression analysis by microarray identified 90 differentially expressed genes in the testis in response to hCG administration, including genes potentially involved in steroidogenesis, progression of spermatogenesis and germ cell maturation and cytoskeletal organization. Our results provide evidence for the first time that key genes involved in the regulation of steroid production and spermatogenesis in the Senegalese sole testis are under gonadotropic control.
2010-12-02 | GSE25733 | GEO
Project description:Tomàs-Gamisans2016 - Genome-Scale Metabolic Model of Pichia pastoris (version 2)
Note:
This is iMT1026v2, an update of the original iMT1026 Pichia pastoris' genome-scale metabolic model PMID:26812499. iMT1026v2 has incorporated reactions involved in xylulose-monophosphate cycle of methanol assimilation pathway described in Rußmayer et al., (2015); additionally 9 reactions were modified fixing wrong metabolite assignments.
This model is described in the article:
Integration and Validation of the Genome-Scale Metabolic Models of Pichia pastoris: A Comprehensive Update of Protein Glycosylation Pathways, Lipid and Energy Metabolism.
Tomàs-Gamisans M, Ferrer P, Albiol J
PLoS One. 2016 Jan 26;11(1):e0148031.
Abstract:
MOTIVATION:
Genome-scale metabolic models (GEMs) are tools that allow predicting a phenotype from a genotype under certain environmental conditions. GEMs have been developed in the last ten years for a broad range of organisms, and are used for multiple purposes such as discovering new properties of metabolic networks, predicting new targets for metabolic engineering, as well as optimizing the cultivation conditions for biochemicals or recombinant protein production. Pichia pastoris is one of the most widely used organisms for heterologous protein expression. There are different GEMs for this methylotrophic yeast of which the most relevant and complete in the published literature are iPP668, PpaMBEL1254 and iLC915. However, these three models differ regarding certain pathways, terminology for metabolites and reactions and annotations. Moreover, GEMs for some species are typically built based on the reconstructed models of related model organisms. In these cases, some organism-specific pathways could be missing or misrepresented.
RESULTS:
In order to provide an updated and more comprehensive GEM for P. pastoris, we have reconstructed and validated a consensus model integrating and merging all three existing models. In this step a comprehensive review and integration of the metabolic pathways included in each one of these three versions was performed. In addition, the resulting iMT1026 model includes a new description of some metabolic processes. Particularly new information described in recently published literature is included, mainly related to fatty acid and sphingolipid metabolism, glycosylation and cell energetics. Finally the reconstructed model was tested and validated, by comparing the results of the simulations with available empirical physiological datasets results obtained from a wide range of experimental conditions, such as different carbon sources, distinct oxygen availability conditions, as well as producing of two different recombinant proteins. In these simulations, the iMT1026 model has shown a better performance than the previous existing models.
This model is hosted on
BioModels Database
and identified by:
MODEL1508040001.
To cite BioModels Database, please use:
BioModels:
Content, Features, Functionality and Use.
To the extent possible under law, all copyright and related or
neighbouring rights to this encoded model have been dedicated to
the public domain worldwide. Please refer to
CC0
Public Domain Dedication for more information.
2016-02-10 | MODEL1508040001 | BioModels
Project description:Heterogeneity of protein glycosylation poses great challenge for analysis that is key to un-puzzle systems glycobiology in diseases. Resolving this conundrum requires global enrichment of glycopeptides for identification and quantitation. To this aim, hydrophilic interaction chromatography (HILIC) has been proved to be an effective approach to enrich N-linked glycopeptides (N-glycopeptides). However, its effectiveness to enrich O-linked glycopeptides (O-glycopeptides) and isobaric labelled glycopeptides remains unclear. Here, we studied three different cartridges in HILIC mode. It was found that removal of N-glycosylation prior to enrichment improved identification of O-glycopeptides. It was noted that increased yield and number of glycosylation identification were seen when using cartridges having materials for strong anion exchange (SAX) chromatography. Remarkably, O-glycopeptides were found to be selectively enriched by HILIC with further improved enrichment being seen when Retain AX cartridge being used. Of particular note, isobaric labelled glycopeptides could be readily enriched by SAX cartridges in HILIC mode to enable quantitative glycoproteomics. It is anticipated that the use of SAX cartridges will facilitate broad applications of qualitative and quantitative glycoproteomics in diseases.
2018-07-02 | MSV000082566 | MassIVE