Browse
Submit Data
Databases
API
Help

Dataset Information

17 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Semi-quantitative measurement of a specific glycoform using a DNA-tagged antibody and lectin affinity chromatography for glyco-biomarker development.

ABSTRACT: Aberrant glycosylation-targeted disease biomarker development is based on cumulative evidence that certain glycoforms are mass-produced in a disease-specific manner. However, the development process has been hampered by the absence of an efficient validation method based on a sensitive and multiplexed platform. In particular, ELISA-based analytical tools are not adequate for this purpose, mainly because of the presence of a pair of N-glycans of IgG-type antibodies. To overcome the associated hurdles in this study, antibodies were tagged with oligonucleotides with T7 promoter and then allowed to form a complex with corresponding antigens. An antibody-bound specific glycoform was isolated by lectin chromatography and quantitatively measured on a DNA microarray chip following production of fluorescent RNA by T7-trascription. This tool ensured measurement of targeted glycoforms of multiple biomarkers with high sensitivity and multiplexity. This analytical method was applied to an in vitro diagnostic multivariate index assay where a panel of hepatocellular carcinoma (HCC) biomarkers comprising alpha-fetoprotein, hemopexin, and alpha-2-macroglobulin (A2M) was examined in terms of the serum level and their fuco-fractions. The results indicated that the tests using the multiplexed fuco-biomarkers provided improved discriminatory power between non- hepatocellular carcinoma and hepatocellular carcinoma subjects compared with the alpha-fetoprotein level or fuco-alpha-fetoprotein test alone. The developed method is expected to facilitate the validation of disease-specific glycan biomarker candidates.

SUBMITTER: Lee JH

PROVIDER: S-EPMC4349995 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Multi-lectin Affinity Chromatography and Quantitative Proteomic Analysis Reveal Differential Glycoform Levels between Prostate Cancer and Benign Prostatic Hyperplasia Sera.

Project description:Currently prostate-specific antigen is used for prostate cancer (PCa) screening, however it lacks the necessary specificity for differentiating PCa from other diseases of the prostate such as benign prostatic hyperplasia (BPH), presenting a clinical need to distinguish these cases at the molecular level. Protein glycosylation plays an important role in a number of cellular processes involved in neoplastic progression and is aberrant in PCa. In this study, we systematically interrogate the alterations in the circulating levels of hundreds of serum proteins and their glycoforms in PCa and BPH samples using multi-lectin affinity chromatography and quantitative mass spectrometry-based proteomics. Specific lectins (AAL, PHA-L and PHA-E) were used to target and chromatographically separate core-fucosylated and highly-branched protein glycoforms for analysis, as differential expression of these glycan types have been previously associated with PCa. Global levels of CD5L, CFP, C8A, BST1, and C7 were significantly increased in the PCa samples. Notable glycoform-specific alterations between BPH and PCa were identified among proteins CD163, C4A, and ATRN in the PHA-L/E fraction and among C4BPB and AZGP1 glycoforms in the AAL fraction. Despite these modest differences, substantial similarities in glycoproteomic profiles were observed between PCa and BPH sera.

| S-EPMC5916935 | biostudies-literature

Glycoform-resolved Fc?RIIIa affinity chromatography-mass spectrometry.

Project description:Determination of the impact of individual antibody glycoforms on Fc?RIIIa affinity, and consequently antibody-dependent cell-mediated cytotoxicity (ADCC) previously required high purity glycoengineering. We hyphenated Fc?RIIIa affinity chromatography to mass spectrometry, which allowed direct affinity comparison of glycoforms of intact monoclonal antibodies. The approach enabled reproduction and refinement of known glycosylation effects, and insights on afucosylation pairing as well as on low-abundant, unstudied glycoforms. Our method greatly improves the understanding of individual glycoform structure-function relationships. Thus, it is highly relevant for assessing Fc-glycosylation critical quality attributes related to ADCC.

| S-EPMC6748599 | biostudies-literature

A Quantitative Microtiter Assay for Sialylated Glycoform Analyses Using Lectin Complexes.

Project description:Fidelity of glycan structures is a key requirement for biotherapeutics, with carbohydrates playing an important role for therapeutic efficacy. Comprehensive glycan profiling techniques such as liquid chromatography (LC) and mass spectrometry (MS), while providing detailed description of glycan structures, require glycan cleavage, labeling, and paradigms to deconvolute the considerable data sets they generate. On the other hand, lectins as probes on microarrays have recently been used in orthogonal approaches for in situ glycoprofiling but require analyte labeling to take advantage of the capabilities of automated microarray readers and data analysis they afford. Herein, we describe a lectin-based microtiter assay (lectin-enzyme-linked immunosorbent assay [ELISA]) to quantify terminal glycan moieties, applicable to in vitro and in-cell glycan-engineered Fc proteins as well as intact IgGs from intravenous immunoglobulin (IVIG), a blood product containing pooled polyvalent IgG antibodies extracted from plasma from healthy human donors. We corroborate our findings with industry-standard LC-MS profiling. This "customizable" ELISA juxtaposes readouts from multiple lectins, focusing on a subset of glycoforms, and provides the ability to discern single- versus dual-arm glycosylation while defining levels of epitopes at sensitivities comparable to MS. Extendable to other biologics, this ELISA can be used stand-alone or complementary to MS for quantitative glycan analysis.

| S-EPMC4512520 | biostudies-literature

Efficacy of glycoprotein enrichment by microscale lectin affinity chromatography.

Project description:Reproducible and efficient affinity enrichment is increasingly viewed as an essential step in many investigations leading to the discovery of new biomarkers. In this work, we have evaluated the repeatability of lectin enrichment of glycoproteins from human blood serum through both qualitative and quantitative proteomic approaches. In a comprehensive evaluation of lectin binding, we have performed 30 separate microscale lectin affinity chromatography experiments, followed by a conventional sample purification, and LC-MS/MS analysis of the enriched glycoproteins. Two lectin affinity matrixes, both with Con A lectin, immobilized to the same solid support but differing in the amount of immobilized lectin, were investigated to characterize their binding properties. Both qualitative and quantitative data indicate acceptable repeatability and binding efficiency for the lectin materials received from two different commercial sources.

| S-EPMC3658453 | biostudies-literature

Lectin affinity chromatography and quantitative proteomic analysis reveal that galectin-3 is associated with metastasis in nasopharyngeal carcinoma.

Project description:Nasopharyngeal carcinoma (NPC) is a serious cancer in East and Southeast Asia. Patients are often diagnosed at advanced stages, rendering treatment failure due to high potential of metastasis. This study identified lectin-binding glycoproteins with a potential role in NPC metastasis. Cell lysate and culture medium in highly metastatic 5-8F, and lowly-metastatic 6-10B NPC cell lines were fractionated by ConA- and WGA-affinity chromatography, and subjected to GeLC-MS/MS. A total of 232 and 197 proteins were identified in ConA-enriched fraction of 5-8F and 6-10B cell lysates respectively. In WGA-enriched fraction, 65 and 164 proteins were found in 5-8F and 6-10B cell lysates respectively. Proteins identified in culture medium for both cell lines were 223 and 85 for ConA-enriched fraction, and 94 and 124 for WGA-enriched fraction from 5-8F and 6-10B respectively. Differentially expressed proteins were functionally categorized into cell-cell adhesion, extracellular matrix, glycolysis, protein homeostasis and/or glycosylation enzymes, and lipid metabolism. Interestingly, Galectin-3 (Gal-3) was highly expressed in 5-8F cells but was lowly expressed in 6-10B cells. The Gal-3 knockdown in 5-8F cells, Gal-3 overexpression in 6-10B cells and treatment with Gal-3 inhibitor revealed that Gal-3 was responsible for metastatic phenotypes including adhesion, migration and invasion. So Galectin-3 may serve as a potential target for NPC therapeutic interventions.

| S-EPMC7536187 | biostudies-literature

Characterization of murine brain membrane glycoproteins by detergent assisted lectin affinity chromatography.

Project description:Membrane glycoproteins play vital roles in many fundamental physiological and pathophysiological processes in the central nervous system and represent important targets for pharmaceuticals and biomarker discovery. However, their isolation and characterization have been greatly limited. Lectin affinity chromatography (LAC) has evolved as a powerful method to enrich glycoproteins in biofluid and cell/tissue lysate. However, its use in the hydrophobic fraction of the samples has rarely been explored. In this study, we have conducted a systematic investigation on the lectin binding efficiency in the presence of four commonly used detergents. We have found that, under certain concentrations, detergents can minimize nonspecific binding and facilitate the elution of hydrophobic glycoproteins. With the detergent assisted lectin affinity chromatography (DALAC), a total of 1491 proteins were identified with low numbers of false positives from two lectins. Proteins (699) were identified with at least two unique peptides, of which 219 are membrane glycoproteins. Compared to the traditional methods, the DALAC approach significantly increased the recovery of plasma membrane and glycoproteins. NP-40 is recommended as a well rounded detergent for DALAC, but the conditions for enriching certain target proteins need to be empirically determined. This study represents the first global identification of the murine brain glycoproteome.

| S-EPMC2930182 | biostudies-literature

Immobilized metal affinity chromatography co-purifies TGF-?1 with histidine-tagged recombinant extracellular proteins.

Project description:Extracellular recombinant proteins are commonly produced using HEK293 cells as histidine-tagged proteins facilitating purification by immobilized metal affinity chromatography (IMAC). Based on gel analyses, this one-step purification typically produces proteins of high purity. Here, we analyzed the presence of TGF-?1 in such IMAC purifications using recombinant extracellular fibrillin-1 fragments as examples. Analysis of various purified recombinant fibrillin-1 fragments by ELISA consistently revealed the presence of picomolar concentrations of active and latent TGF-?1, but not of BMP-2. These quantities of TGF-?1 were not detectable by Western blotting and mass spectrometry. However, the amounts of TGF-?1 were sufficient to consistently trigger Smad2 phosphorylation in fibroblasts. The purification mechanism was analyzed to determine whether the presence of TGF-?1 in these protein preparations represents a specific or non-specific co-purification of TGF-?1 with fibrillin-1 fragments. Control purifications using conditioned medium from non-transfected 293 cells yielded similar amounts of TGF-?1 after IMAC. IMAC of purified TGF-?1 and the latency associated peptide showed that these proteins bound to the immobilized nickel ions. These data clearly demonstrate that TGF-?1 was co-purified by specific interactions with nickel, and not by specific interactions with fibrillin-1 fragments. Among various chromatographic methods tested for their ability to eliminate TGF-?1 from fibrillin-1 preparations, gel filtration under high salt conditions was highly effective. As various recombinant extracellular proteins purified in this fashion are frequently used for experiments that can be influenced by the presence of TGF-?1, these findings have far-reaching implications for the required chromatographic schemes and quality controls.

| S-EPMC3485342 | biostudies-literature

The Lectin Frontier Database (LfDB), and data generation based on frontal affinity chromatography.

Project description:Lectins are a large group of carbohydrate-binding proteins, having been shown to comprise at least 48 protein scaffolds or protein family entries. They occur ubiquitously in living organisms-from humans to microorganisms, including viruses-and while their functions are yet to be fully elucidated, their main underlying actions are thought to mediate cell-cell and cell-glycoconjugate interactions, which play important roles in an extensive range of biological processes. The basic feature of each lectin's function resides in its specific sugar-binding properties. In this regard, it is beneficial for researchers to have access to fundamental information about the detailed oligosaccharide specificities of diverse lectins. In this review, the authors describe a publicly available lectin database named "Lectin frontier DataBase (LfDB)", which undertakes the continuous publication and updating of comprehensive data for lectin-standard oligosaccharide interactions in terms of dissociation constants (Kd's). For Kd determination, an advanced system of frontal affinity chromatography (FAC) is used, with which quantitative datasets of interactions between immobilized lectins and >100 fluorescently labeled standard glycans have been generated. The FAC system is unique in its clear principle, simple procedure and high sensitivity, with an increasing number (>67) of associated publications that attest to its reliability. Thus, LfDB, is expected to play an essential role in lectin research, not only in basic but also in applied fields of glycoscience.

| S-EPMC6272529 | biostudies-literature

Analysis of Tagged Proteins Using Tandem Affinity-Buffer Exchange Chromatography Online with Native Mass Spectrometry.

Project description:Protein overexpression and purification are critical for in vitro structure-function characterization studies. However, some proteins are difficult to express in heterologous systems due to host-related (e.g., codon usage, translation rate) and/or protein-specific (e.g., toxicity, aggregation) challenges. Therefore, it is often necessary to test multiple overexpression and purification conditions to maximize the yield of functional protein, particularly for resource-heavy downstream applications (e.g., biocatalysts, tertiary structure determination, biotherapeutics). Here, we describe an automatable liquid chromatography-mass spectrometry-based method for direct analysis of target proteins in cell lysates. This approach is facilitated by coupling immobilized metal affinity chromatography (IMAC), which leverages engineered poly-histidine tags in proteins of interest, with size exclusion-based online buffer exchange (OBE) and native mass spectrometry (nMS). While we illustrate a proof of concept here using relatively straightforward examples, the use of IMAC-OBE-nMS to optimize conditions for large-scale protein production may become invaluable for expediting structural biology and biotherapeutic initiatives.

| S-EPMC9080447 | biostudies-literature

Quantitative analysis of fucosylated glycoproteins by immobilized lectin-affinity fluorescent labeling.

Project description:Human biofluids are often used to discover disease-specific glycosylation, since abnormal changes in protein glycosylation can discern physiopathological states. Highly glycosylated proteins in biofluids make it possible to identify disease signatures. Glycoproteomic studies on saliva glycoproteins showed that fucosylation was significantly increased during tumorigenesis and that glycoproteins became hyperfucosylated in lung metastases, and tumor stage is associated with fucosylation. Quantification of salivary fucosylation can be achieved by mass spectrometric analysis of fucosylated glycoproteins or fucosylated glycans; however, the use of mass spectrometry is non-trivial for clinical practice. Here, we developed a high-throughput quantitative method, lectin-affinity fluorescent labeling quantification (LAFLQ), to quantify fucosylated glycoproteins without relying on mass spectrometry. Lectins with a specific affinity for fucoses are immobilized on the resin and effectively capture fluorescently labeled fucosylated glycoproteins, which are further quantitatively characterized by fluorescence detection in a 96-well plate. Our results demonstrated that serum IgG can be accurately quantified by lectin and fluorescence detection. Quantification in saliva showed significantly higher fucosylation in lung cancer patients compared to healthy controls or other non-cancer diseases, suggesting that this method has the potential to quantify stage-related fucosylation in lung cancer saliva.

| S-EPMC9969232 | 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