Abstract:Fibrinogen (FIB) is a secretory glycoprotein synthesized by hepatocytes that has a key role in blood clotting. Its glycosylation has not been studied in detail and little is known about the biological variability of FIB N-glycosylation, mainly due to the lack of fast, simple, and robust approaches to purify FIB from blood plasma samples. In recent years, customised chromatographic monoliths have been used for a variety of biological applications due to their unique characteristics. Here we describe development… Show more
“…Both protein precipitation and corona enrichment are not as selective as the affinity method; however, the major advantage of the corona enrichment method for glycan profiling compared to the precipitation method is that it could be utilized in different contexts, for different corona protein enrichment by changing the surface chemistry or plasma protein/NP ratio. The affinity method provided good selectivity, and its application in glycan profiling has been demonstrated . However, the cost of the antibodies and the optimization steps would need to be considered in a cohort analysis.…”
Biomolecular
corona formation has emerged as a recurring and important
phenomenon in nanomedicine that has been investigated for potential
applications in disease diagnosis. In this study, we have combined
the “personalized protein corona” with the N-glycosylation
profiling that has recently gained considerable interest in human
plasma biomarker discovery as a powerful early warning diagnostic
and patient stratification tool. We envisioned that the protein corona
formation could be exploited as an enrichment step that is critically
important in both proteomic and proteoglycomic workflows. By using
silica nanoparticles, plasma fibrinogen was enriched to a level in
which its proteomic and glycomic “fingerprints” could
be traced with confidence. Despite being a more simplified glycan
profile compared to full plasma, the corona glycan profile revealed
a fibrinogen-derived glycan peak that was found to potentially distinguish
lung cancer patients from controls in a pilot study.
“…Both protein precipitation and corona enrichment are not as selective as the affinity method; however, the major advantage of the corona enrichment method for glycan profiling compared to the precipitation method is that it could be utilized in different contexts, for different corona protein enrichment by changing the surface chemistry or plasma protein/NP ratio. The affinity method provided good selectivity, and its application in glycan profiling has been demonstrated . However, the cost of the antibodies and the optimization steps would need to be considered in a cohort analysis.…”
Biomolecular
corona formation has emerged as a recurring and important
phenomenon in nanomedicine that has been investigated for potential
applications in disease diagnosis. In this study, we have combined
the “personalized protein corona” with the N-glycosylation
profiling that has recently gained considerable interest in human
plasma biomarker discovery as a powerful early warning diagnostic
and patient stratification tool. We envisioned that the protein corona
formation could be exploited as an enrichment step that is critically
important in both proteomic and proteoglycomic workflows. By using
silica nanoparticles, plasma fibrinogen was enriched to a level in
which its proteomic and glycomic “fingerprints” could
be traced with confidence. Despite being a more simplified glycan
profile compared to full plasma, the corona glycan profile revealed
a fibrinogen-derived glycan peak that was found to potentially distinguish
lung cancer patients from controls in a pilot study.
“…Since high flow rates can easily be applied and analysis time can be very short, monoliths are perfect supports for HTP screening . A semi‐HTP isolation of human fibrinogen using monolithic supports with immobilized monoclonal antihuman fibrinogen antibodies was successfully performed . Another group used monoliths for HTP isolation of transferrin from human plasma by use of antitransferrin monoclonal antibodies immobilized on monoliths .…”
Posttranslational modifications of immunoglobulins have been a topic of great interest and have been repeatedly reported as a major factor in disease pathology. Cost-effective, reproducible, and high-throughput (HTP) isolation of immunoglobulins from human serum is vital for studying the changes in protein structure and the following understanding of disease development. Although there are many methods for the isolation of specific immunoglobulin classes, only a few of them are applicable for isolation of all subtypes and variants. Here, we present the development of a scheme for fast and simultaneous affinity purification of α (A), γ (G), and μ (M) immunoglobulins from human serum through affinity monolith chromatography. Affinity-based monolithic columns with immobilized protein A, G, or L were used for antibody isolation. Monolithic stationary phases have a high surface accessibility of binding sites, large flow-through channels, and can be operated at high flow rates, making them the ideal supports for HTP isolation of biopolymers. The presented method can be used for HTP screening of human serum in order to simultaneously isolate all three above-mentioned immunoglobulins and determine their concentration and changes in their glycosylation pattern as potential prognostic and diagnostic disease biomarkers.
“…6) [265]. Several commercial organic monoliths were employed with IAC and anti‐human fibrinogen antibodies for the isolation of fibrinogen from human blood samples [266]. CIM disks were used to immobilize protein G, which was then used to adsorb antibodies for the capture of ammodytoxins (i.e., the most toxic components of the snake venom) and their interacting protein partners from snake venom [267].…”
Affinity monolith chromatography (AMC) is a liquid chromatographic technique that utilizes a monolithic support with a biological ligand or related binding agent to isolate, enrich, or detect a target analyte in a complex matrix. The target‐specific interaction exhibited by the binding agents makes AMC attractive for the separation or detection of a wide range of compounds. This article will review the basic principles of AMC and recent developments in this field. The supports used in AMC will be discussed, including organic, inorganic, hybrid, carbohydrate, and cryogel monoliths. Schemes for attaching binding agents to these monoliths will be examined as well, such as covalent immobilization, biospecific adsorption, entrapment, molecular imprinting, and coordination methods. An overview will then be given of binding agents that have recently been used in AMC, along with their applications. These applications will include bioaffinity chromatography, immunoaffinity chromatography, immobilized metal‐ion affinity chromatography, and dye‐ligand or biomimetic affinity chromatography. The use of AMC in chiral separations and biointeraction studies will also be discussed.
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