Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Structural Identification of a Non-Glycosylated Variant at Ser126 for O-Glycosylation Site from EPO BRP, Human Recombinant Erythropoietin by LC/MS Analysis.

ABSTRACT: A variant peak was detected in the analysis of RP-HPLC of rHu-EPO, which has about 7% relative content. Fractions of the main and the variant peaks were pooled separately and further analyzed to identify the molecular structure of the variant peak. Total mass analysis for each peak fraction using ESI-TOF MS shows differences in molecular mass. The fraction of the main peak tends to result in higher molecular masses than the fraction of the variant. The detected masses for the variant are about 600-1000 Da smaller than those for the main peak. Peptide mapping analysis for each peak fraction using Asp-N and Glu-C shows differences in O-glycopeptide profiles at Ser126. The O-glycopeptides were not detected in the fraction of the variant. It is concluded that the variant peak is non-O-glycosylated rHu-EPO and the main peak is fully O-glycosylated rHu-EPO at Ser126.

SUBMITTER: Byeon J

PROVIDER: S-EPMC4469907 | biostudies-other | 2015 Jun

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Publications

Structural Identification of a Non-Glycosylated Variant at Ser126 for O-Glycosylation Site from EPO BRP, Human Recombinant Erythropoietin by LC/MS Analysis.

Byeon Jaehee J Lim Yu-Ri YR Kim Hyong-Ha HH Suh Jung-Keun JK

Molecules and cells 20150527 6

A variant peak was detected in the analysis of RP-HPLC of rHu-EPO, which has about 7% relative content. Fractions of the main and the variant peaks were pooled separately and further analyzed to identify the molecular structure of the variant peak. Total mass analysis for each peak fraction using ESI-TOF MS shows differences in molecular mass. The fraction of the main peak tends to result in higher molecular masses than the fraction of the variant. The detected masses for the variant are about 6 ...[more]

PMID: 26013384

Similar Datasets

Structural identification of modified amino acids on the interface between EPO and its receptor from EPO BRP, human recombinant erythropoietin by LC/MS analysis.

Project description:Protein modifications of recombinant pharmaceuticals have been observed both in vitro and in vivo. These modifications may result in lower efficacy, as well as bioavailability changes and antigenicity among the protein pharmaceuticals. Therefore, the contents of modification should be monitored for the quality and efficacy of protein pharmaceuticals. The interface of EPO and its receptor was visualized, and potential amino acids interacting on the interface were also listed. Two different types of modifications on the interface were identified in the preparation of rHu-EPO BRP. A UPLC/Q-TOF MS method was used to evaluate the modification at those variants. The modification of the oxidized variant was localized on the Met54 and the deamidated variants were localized on the Asn47 and Asn147. The extent of oxidation at Met54 was 3.0% and those of deamidation at Asn47 and Asn147 were 2.9% and 4.8%, respectively.

| S-EPMC4255102 | biostudies-literature

Mass spectrometry (LC-MS/MS) site-mapping of N-glycosylated membrane proteins for breast cancer biomarkers.

Project description:Cancer cell membrane proteins are released into the plasma/serum by exterior protein cleavage, membrane sloughing, cellular secretion or cell lysis, and represent promising candidates for interrogation. Because many known disease biomarkers are both glycoproteins and membrane bound, we chose the hydrazide method to specifically target, enrich, and identify glycosylated proteins from breast cancer cell membrane fractions using the LTQ Orbitrap mass spectrometer. Our initial goal was to select membrane proteins from breast cancer cell lines and then to use the hydrazide method to identify the N-linked proteome as a prelude to evaluation of plasma/serum proteins from cancer patients. A combination of steps facilitated identification of the glycopeptides and also defined the glycosylation sites. In MCF-7, MDA-MB-453 and MDA-MB-468 cell membrane fractions, use of the hydrazide method facilitated an initial enrichment and site mapping of 27 N-linked glycosylation sites in 25 different proteins. However, only three N-linked glycosylated proteins, galectin-3 binding protein, lysosome associated membrane glycoprotein 1, and oxygen regulated protein, were identified in all three breast cancer cell lines. In addition, MCF-7 cells shared an additional 3 proteins with MDA-MB-453. Interestingly, the hydrazide method isolated a number of other N-linked glycoproteins also known to be involved in breast cancer, including epidermal growth factor receptor (EGFR), CD44, and the breast cancer 1, and early onset isoform 1 (BRCA1) biomarker. Analyzing the N-glycoproteins from membranes of breast cancer cell lines highlights the usefulness of the procedure for generating a practical set of potential biomarkers.

| S-EPMC2761014 | biostudies-literature

Characterization of the Glycosylation Site of Human PSA Prompted by Missense Mutation using LC-MS/MS.

Project description:Prostate specific antigen (PSA) is currently used as a diagnostic biomarker for prostate cancer. It is a glycoprotein possessing a single glycosylation site at N69. During our previous study of PSA N69 glycosylation, additional glycopeptides were observed in the PSA sample that were not previously reported and did not match glycopeptides of impure glycoproteins existing in the sample. This extra glycosylation site of PSA is associated with a mutation in KLK3 genes. Among single nucleotide polymorphisms (SNPs) of KLKs families, the rs61752561 in KLK3 genes is an unusual missense mutation resulting in the conversion of D102 to N in PSA amino acid sequence. Accordingly, a new N-linked glycosylation site is created with an N102MS motif. Here we report the first qualitative and quantitative glycoproteomic study of PSA N102 glycosylation site by LC-MS/MS. We successfully applied tandem MS to verify the amino acid sequence possessing N102 glycosylation site and associated glycoforms of PSA samples acquired from different suppliers. Among the three PSA samples, HexNAc2Hex5 was the predominant glycoform at N102, while HexNAc4Hex5Fuc1NeuAc1 or HexNAc4Hex5Fuc1NeuAc2 was the primary glycoforms at N69. D102 is the first amino acid of "kallikrein loop", which is close to a zinc-binding site and catalytic triad. The different glycosylation of N102 relative to N69 might be influenced by the close vicinity of N102 to these functional sites and steric hindrance.

| S-EPMC4866810 | biostudies-literature

Absolute quantitation of glycosylation site occupancy using isotopically labeled standards and LC-MS.

Project description:N-linked glycans are required to maintain appropriate biological functions on proteins. Underglycosylation leads to many diseases in plants and animals; therefore, characterizing the extent of glycosylation on proteins is an important step in understanding, diagnosing, and treating diseases. To determine the glycosylation site occupancy, protein N-glycosidase F (PNGase F) is typically used to detach the glycan from the protein, during which the formerly glycosylated asparagine undergoes deamidation to become an aspartic acid. By comparing the abundance of the resulting peptide containing aspartic acid against the one containing non-glycosylated asparagine, the glycosylation site occupancy can be evaluated. However, this approach can give inaccurate results when spontaneous chemical deamidation of the non-glycosylated asparagine occurs. To overcome this limitation, we developed a new method to measure the glycosylation site occupancy that does not rely on converting glycosylated peptides to their deglycosylated forms. Specifically, the overall protein concentration and the non-glycosylated portion of the protein are quantified simultaneously by using heavy isotope-labeled internal standards coupled with LC-MS analysis, and the extent of site occupancy is accurately determined. The efficacy of the method was demonstrated by quantifying the occupancy of a glycosylation site on bovine fetuin. The developed method is the first work that measures the glycosylation site occupancy without using PNGase F, and it can be done in parallel with glycopeptide analysis because the glycan remains intact throughout the workflow.

| S-EPMC4458369 | biostudies-literature

Erythropoietin (EPO) in acute kidney injury.

Project description:Erythropoietin (EPO) is a 30.4 kDa glycoprotein produced by the kidney, and is mostly well-known for its physiological function in regulating red blood cell production in the bone marrow. Accumulating evidence, however, suggests that EPO has additional organ protective effects, which may be useful in the prevention or treatment of acute kidney injury. These protective mechanisms are multifactorial in nature and include inhibition of apoptotic cell death, stimulation of cellular regeneration, inhibition of deleterious pathways, and promotion of recovery.In this article, we review the physiology of EPO, assess previous work that supports the role of EPO as a general tissue protective agent, and explain the mechanisms by which it may achieve this tissue protective effect. We then focus on experimental and clinical data that suggest that EPO has a kidney protective effect.

| S-EPMC3159901 | biostudies-literature

The analysis of alpha-1-antitrypsin glycosylation with direct LC-MS/MS.

Project description:A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based methodology has been developed to differentiate core- and antennary-fucosylated glycosylation of glycopeptides. Both the glycosylation sites (heterogeneity) and multiple possible glycan occupancy at each site (microheterogeneity) can be resolved via intact glycopeptide analysis. The serum glycoprotein alpha-1-antitrypsin (A1AT) which contains both core- and antennary-fucosylated glycosites was used in this study. Sialidase was used to remove the sialic acids in order to simplify the glycosylation microheterogeneity and to enhance the MS signal of glycopeptides with similar glycan structures. β1-3,4 galactosidase was used to differentiate core- and antennary-fucosylation. In-source dissociation was found to severely affect the identification and quantification of glycopeptides with low abundance glycan modification. The settings of the mass spectrometer were therefore optimized to minimize the in-source dissociation. A three-step mass spectrometry fragmentation strategy was used for glycopeptide identification, facilitated by pGlyco software annotation and manual checking. The collision energy used for initial glycopeptide fragmentation was found to be crucial for improved detection of oxonium ions and better selection of Y1 ion (peptide+GlcNAc). Structural assignments revealed that all three glycosylation sites of A1AT glycopeptides contain complex N-glycan structures: site Asn70 contains biantennary glycans without fucosylation; site Asn107 contains bi-, tri- and tetra-antennary glycans with both core- and antennary-fucosylation; site Asn271 contains bi- and tri-antennary glycans with both core- and antennary-fucosylation. The relative intensity of core- and antennary-fucosylation on Asn107 was similar to that of the A1AT protein indicating that the glycosylation level of Asn107 is much larger than the other two sites.

| S-EPMC6077116 | biostudies-literature

Genetic variants of erythropoietin (EPO) and EPO receptor genes in familial erythrocytosis.

Project description:ObjectivesErythrocytosis is characterized by the expansion of erythrocyte compartment including elevated red blood cell number, hematocrit, and hemoglobin content. Familial erythrocytosis (FE) is a congenital disorder with different genetic background. Type 1 FE is primary FE caused by mutation in erythropoietin receptor gene (EPOR). Type 2-5 FE are secondary FEs caused by mutations of genes involved in oxygen sensing pathway important for erythropoietin (EPO) regulation. In the present study, we summarized associations between EPOR and EPO gene variations with development of FE and searched for genetic variants located within regulatory regions.MethodsPublications reporting EPOR and EPO sequence variants associated with FE or clinical features of erythrocytosis were retrieved from PubMed and WoS. In silico, sequence reanalysis was performed using Ensembl genomic browser, release 89 to screen for variants located within regulatory regions.ResultsTo date, 28 variants of the EPOR and seven variants of the EPO gene have been associated with erythrocytosis or upper hematocrit. Sequence variants were also found to be present within regulatory regions.ConclusionsRole of variants in regulatory regions of the EPO gene should be further investigated.

| S-EPMC7379665 | biostudies-literature

Affinity purification of erythropoietin from cell culture supernatant combined with MALDI-TOF-MS analysis of erythropoietin N-glycosylation.

Project description:Erythropoietin (EPO) is a heavily glycosylated hormone whose recombinant forms are used for treatment of anaemia. EPO glycosylation is important for its pharmacological properties. An analytical workflow, which can determine EPO glycosylation in an accurate and high-throughput fashion from cell culture supernatant (CCS) in approximately 24?h, offers the possibility to follow changes during production. To address this challenge, we present a complete workflow consisting of protein purification, glycan release, sialic acid derivatization, solid phase extraction, matrix-assisted laser desorption/ionization - mass spectrometry (MALDI-MS) analysis and MassyTools data processing. EPO purification from CCS by anti-EPO antibody coupled Sepharose beads yielded excellent purity with acceptable recovery and was free of glycoform bias. Glycosylation profiles obtained by MALDI-MS were highly comparable to those obtained with an established capillary gel electrophoresis-laser induced fluorescence method. Our method delivers accurate results for the analysis of changes of important glycosylation parameters, such as sialylation and number of N-acetyllactosamine units, for the time course of a fermentation. We could resolve differences in glycosylation between several CCS samples.

| S-EPMC5509673 | biostudies-literature

Sequential Analysis of the N/O-Glycosylation of Heavily Glycosylated HIV-1 gp120 Using EThcD-sceHCD-MS/MS.

Project description:Deciphering the glycosylation of the viral envelope (Env) glycoprotein is critical for evaluating viral escape from the host's immune response and developing vaccines and antiviral drugs. However, it is still challenging to precisely decode the site-specific glycosylation characteristics of the highly glycosylated Env proteins, although glycoproteomics have made significant advances in mass spectrometry techniques and data analysis tools. Here, we present a hybrid dissociation technique, EThcD-sceHCD, by combining electron transfer/higher-energy collisional dissociation (EThcD) and stepped collision energy/higher-energy collisional dissociation (sceHCD) into a sequential glycoproteomic workflow. Following this scheme, we characterized site-specific N/O-glycosylation of the human immunodeficiency virus type 1 (HIV-1) Env protein gp120. The EThcD-sceHCD method increased the number of identified glycopeptides when compared with EThcD, while producing more comprehensive fragment ions than sceHCD for site-specific glycosylation analysis, especially for accurate O-glycosite assignment. Finally, eighteen N-glycosites and five O-glycosites with attached glycans were assigned unambiguously from heavily glycosylated gp120. These results indicate that our workflow can achieve improved performance for analysis of the N/O-glycosylation of a highly glycosylated protein containing numerous potential glycosites in one process. Knowledge of the glycosylation landscape of the Env glycoprotein will be useful for understanding of HIV-1 infection and development of vaccines and drugs.

| S-EPMC8567067 | biostudies-literature

Expression and analysis of the glycosylation properties of recombinant human erythropoietin expressed in Pichia pastoris.

Project description:The Pichia pastoris expression system was used to produce recombinant human erythropoietin, a protein synthesized by the adult kidney and responsible for the regulation of red blood cell production. The entire recombinant human erythropoietin (rhEPO) gene was constructed using the Splicing by Overlap Extension by PCR (SOE-PCR) technique, cloned and expressed through the secretory pathway of the Pichia expression system. Recombinant erythropoietin was successfully expressed in P. pastoris. The estimated molecular mass of the expressed protein ranged from 32 kDa to 75 kDa, with the variation in size being attributed to the presence of rhEPO glycosylation analogs. A crude functional analysis of the soluble proteins showed that all of the forms were active in vivo.

| S-EPMC3168189 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data