The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
David S. Hage - One of the best experts on this subject based on the ideXlab platform.
-
glycoprotein analysis using lectin Microcolumns and capillary electrophoresis characterization of alpha1 acid glycoprotein by combined separation methods
Journal of Chromatography B, 2021Co-Authors: Chenhua Zhang, Katherine N Schumacher, Eric D Dodds, David S. HageAbstract:Abstract Separations based on combinations of 2.1 mm I.D. high-performance affinity Microcolumns and capillary electrophoresis were developed and used to characterize the glycoforms of an intact glycoprotein. Human alpha1-acid glycoprotein (AGP) was used as a model analyte due to its heterogeneous glycosylation resulting from variations in its degree of branching, fucosylation, and number of sialic acids. Three separation formats were examined based on Microcolumns that contained the lectins concanavalin A (Con A) or Aleuria aurantia lectin (AAL). These Microcolumns were used with one another or in combination with capillary electrophoresis. N-Glycan analysis of the non-retained and retained AGP fractions was carried out by using PNGase F digestion and nanoflow electrospray ionization mass spectrometry. Con A Microcolumns were found to selectively enrich AGP that contained bi-antennary N-glycans, while AAL Microcolumns retained AGP with fucose-containing N-glycans. Results from these separation methods indicated that fucosylation of the N-linked glycans was more abundant when a high degree of branching was present in AGP. Sialic acid residues were more abundant when higher degrees of branching and more fucose residues were present in AGP. The separation and analysis methods that were developed could be used with relatively small amounts of AGP and can be adapted for use with other intact glycoproteins.
-
studies of binding by sulfonylureas with glyoxal and methylglyoxal modified albumin by immunoextraction using affinity Microcolumns
Journal of Chromatography A, 2021Co-Authors: Elliott Rodriguez, Ryan Matsuda, Pingyang Tao, Ashley G Woolfork, Zuchen Sun, David S. HageAbstract:Abstract Diabetes is characterized by elevated levels of blood glucose, which can result in the modification of serum proteins. The modification of a protein by glucose, or glycation, can also lead to the formation of advanced glycated end-products (AGEs). One protein that can be modified through glycation and AGE formation is human serum albumin (HSA). In this study, immunoextraction based on polyclonal anti-HSA antibodies was used with high-performance affinity Microcolumns to see how AGE-related modifications produced by glyoxal (Go) and methylglyoxal (MGo) affected the binding of HSA to several first- and second-generation sulfonylureas, a class of drugs used to treat type II diabetes and known to bind to HSA. With this approach, it was possible to use a single platform to examine drug interactions with several preparations of HSA. Each applied protein sample could be used over 20–50 experiments, and global affinity constants for most of the examined drugs could be obtained in less than 7.5 min. The binding constants measured for these drugs with normal HSA gave good agreement with global affinities based on the literature. Both Go- and MGo-related modifications at clinically relevant levels were found by this method to create significant changes in the binding by some sulfonylureas with HSA. The global affinities for many of the drugs increased by 1.4-fold or more; gliclazide and tolazamide had no significant change with some preparations of modified HSA, and a small-to-moderate decrease in binding strength was noted for glibenclamide and gliclazide with Go-modified HSA. This approach can be adapted for the study of other drug-protein interactions and alternative modified proteins by altering the antibodies that are employed for immunoextraction and within the affinity microcolumn.
-
development of a microcolumn one site immunometric assay for a protein biomarker analysis of alpha1 acid glycoprotein
Journal of Chromatography A, 2020Co-Authors: Chenhua Zhang, William Clarke, Shae Lott, David S. HageAbstract:Abstract A one-site immunometric assay based on affinity Microcolumns was developed for the analysis of alpha1-acid glycoprotein (AGP) as a model protein biomarker. In this assay, a sample containing AGP was incubated with an excess amount of a labeled binding agent, such as fluorescein-labeled anti-AGP antibodies or Fab fragments. The excess binding agent was removed by passing this mixture through a microcolumn that contained an immobilized form of AGP, while the signal was measured for the binding agent-AGP complex in the non-retained fraction. Theoretical and practical factors were both considered in selecting the concentration of labeled binding agent, the incubation time of this agent with the sample, and the application conditions for this mixture onto the microcolumn. The effects of using various labeling methods and intact antibodies vs Fab fragments were also considered. The final assay was performed with fluorescein-labeled anti-AGP antibodies and a 2.1 mm i.d. × 1.0 cm AGP microcolumn operated at 0.30 mL min−1. This method required only 1 µL of serum or plasma, had a detection limit of 0.63 nM AGP, and gave a potential throughput of 2 min per sample. This assay was used to measure AGP in normal serum and plasma from patients with systemic lupus erythematosus, giving good agreement with the literature and a reference method. The same approach and guidelines can be used to create assays for other protein biomarkers by changing the labeled binding agent and immobilized protein within the microcolumn.
-
peak decay analysis and biointeraction studies of immunoglobulin binding and dissociation on protein g affinity Microcolumns
Methods, 2018Co-Authors: Jeanethe Anguizola, Erika L. Pfaunmiller, Mitchell L. Milanuk, David S. HageAbstract:Protein G can be a valuable binding agent for antibodies and immunoglobulins in methods such as immunosensors, chromatographic-based immunoassays, and immunoaffinity chromatography. This report used the method of peak decay analysis along with frontal analysis and zonal elution studies to characterize the binding, elution and regeneration properties of affinity Microcolumns that contained immobilized protein G. Frontal analysis was employed with rabbit immunoglobulin G (IgG) to characterize the binding capacity of these affinity Microcolumns. Zonal elution experiments looking at the retained peaks for small injections of labeled rabbit IgG were used to optimize the column regeneration conditions. Peak decay analysis was then used to look at the effects of flow rate and elution pH on the release of several types of IgG from the protein G Microcolumns. This approach made it possible to obtain detailed information on the use and behavior of such columns, as could be used in future work to optimize the capture or analysis of IgG and antibodies by such devices. The same approach and tools that were used in this report could also be adapted for work with affinity columns that make use of other supports, binding agents or targets.
-
studies of drug interactions with alpha1 acid glycoprotein by using on line immunoextraction and high performance affinity chromatography
Journal of Chromatography A, 2017Co-Authors: Ryan Matsuda, William Clarke, Chenhua Zhang, Zitha Isingizwe, David S. HageAbstract:Abstract A method that combined on-line immunoextraction with high-performance affinity chromatography was developed to examine the binding of drugs with α 1 -acid glycoprotein (AGP). Affinity Microcolumns containing immobilized polyclonal anti-AGP antibodies were developed that had a capture efficiency of up to 98.4% for AGP and a binding capacity of 0.72 nmol AGP when using a 20 mm × 2.1 mm i.d. microcolumn. These Microcolumns were employed in various formats to examine the binding of drugs to normal AGP and AGP that had been adsorbed from serum samples for patients with systemic lupus erythematosus (SLE). Drugs that were screened in zonal elution experiments for their overall binding to these types of AGP included chlorpromazine, disopyramide, imipramine, propranolol, and warfarin. Most of these drugs showed an increase in their binding to the AGP from SLE serum when compared to normal AGP (i.e., an increase of 13–76%); however, disopyramide gave a 21–25% decrease in retention when the same AGP samples were compared. Frontal analysis was used to further evaluate the binding of disopyramide and imipramine to these forms of AGP. Both drugs gave a good fit to a model that involved a combination of saturable and non-saturable interactions with AGP. Changes in the non-saturable interactions accounted for most of variations seen in the binding of disopyramide and imipramine with the AGP samples. The methods used in this study could be adapted for use in personalized medicine and the study of other proteins or drugs using aqueous mixtures or clinical samples.
Matthias Mann - One of the best experts on this subject based on the ideXlab platform.
-
stop and go extraction tips for matrix assisted laser desorption ionization nanoelectrospray and lc ms sample pretreatment in proteomics
Analytical Chemistry, 2003Co-Authors: Juri Rappsilber, Yasushi Ishihama, Matthias MannAbstract:Proteomics is critically dependent on optimal sample preparation. Particularly, the interface between protein digestion and mass spectrometric analysis has a large influence on the overall quality and sensitivity of the analysis. We here describe a novel procedure in which a very small disk of beads embedded in a Teflon meshwork is placed as a microcolumn into pipet tips. Termed Stage, for STop And Go Extraction, the procedure has been implemented with commercially available material (C18 Empore Disks (3M, Minneapolis, MN)) as frit and separation material. The disk is introduced in a simple and fast process yielding a convenient and completely reliable procedure for the production of self-packed Microcolumns in pipet tips. It is held in place free of obstacles solely by the narrowing tip, ensuring optimized loading and elution of analytes. Five disks are conveniently placed in 1 min, adding 300 μL/...
-
stop and go extraction tips for matrix assisted laser desorption ionization nanoelectrospray and lc ms sample pretreatment in proteomics
Analytical Chemistry, 2003Co-Authors: Juri Rappsilber, Yasushi Ishihama, Matthias MannAbstract:Proteomics is critically dependent on optimal sample preparation. Particularly, the interface between protein digestion and mass spectrometric analysis has a large influence on the overall quality and sensitivity of the analysis. We here describe a novel procedure in which a very small disk of beads embedded in a Teflon meshwork is placed as a microcolumn into pipet tips. Termed Stage, for STop And Go Extraction, the procedure has been implemented with commercially available material (C18 Empore Disks (3M, Minneapolis, MN)) as frit and separation material. The disk is introduced in a simple and fast process yielding a convenient and completely reliable procedure for the production of self-packed Microcolumns in pipet tips. It is held in place free of obstacles solely by the narrowing tip, ensuring optimized loading and elution of analytes. Five disks are conveniently placed in 1 min, adding 300 μL/...
-
Microcolumns with self assembled particle frits for proteomics
Journal of Chromatography A, 2002Co-Authors: Yasushi Ishihama, Juri Rappsilber, Jens S Andersen, Matthias MannAbstract:LC-MS-MS experiments in proteomics are usually performed with packed Microcolumns employing frits or outlets smaller than the particle diameter to retain the packing material. We have developed packed Microcolumns using self-assembled particles (SAPs) as frits that are smaller than the size of the outlet. A five to one ratio of outlet size to particle diameter appears to be the upper maximum. In these situations the particles assembled into an arch over the outlet like the stones in a stone bridge. When 3 microm particles were packed into a tapered column with an 8 microm outlet, two particles bridged the outlet with 0.3 pl dead volume and perfect success rate. In peptide analysis by LC-MS, the peak width at half height was normally less than 6 s, compared to 12 s without SAPs. The LC-MS-MS system provided 37% sequence coverage (21 matched peptides) for a tryptically-digested sample of 10 fmol bovine serum albumin. We also describe application of the SAP principle to make disposable pipette tip columns with short pieces of fused-silica capillary as the outlet.
Ryan Matsuda - One of the best experts on this subject based on the ideXlab platform.
-
studies of binding by sulfonylureas with glyoxal and methylglyoxal modified albumin by immunoextraction using affinity Microcolumns
Journal of Chromatography A, 2021Co-Authors: Elliott Rodriguez, Ryan Matsuda, Pingyang Tao, Ashley G Woolfork, Zuchen Sun, David S. HageAbstract:Abstract Diabetes is characterized by elevated levels of blood glucose, which can result in the modification of serum proteins. The modification of a protein by glucose, or glycation, can also lead to the formation of advanced glycated end-products (AGEs). One protein that can be modified through glycation and AGE formation is human serum albumin (HSA). In this study, immunoextraction based on polyclonal anti-HSA antibodies was used with high-performance affinity Microcolumns to see how AGE-related modifications produced by glyoxal (Go) and methylglyoxal (MGo) affected the binding of HSA to several first- and second-generation sulfonylureas, a class of drugs used to treat type II diabetes and known to bind to HSA. With this approach, it was possible to use a single platform to examine drug interactions with several preparations of HSA. Each applied protein sample could be used over 20–50 experiments, and global affinity constants for most of the examined drugs could be obtained in less than 7.5 min. The binding constants measured for these drugs with normal HSA gave good agreement with global affinities based on the literature. Both Go- and MGo-related modifications at clinically relevant levels were found by this method to create significant changes in the binding by some sulfonylureas with HSA. The global affinities for many of the drugs increased by 1.4-fold or more; gliclazide and tolazamide had no significant change with some preparations of modified HSA, and a small-to-moderate decrease in binding strength was noted for glibenclamide and gliclazide with Go-modified HSA. This approach can be adapted for the study of other drug-protein interactions and alternative modified proteins by altering the antibodies that are employed for immunoextraction and within the affinity microcolumn.
-
studies of drug interactions with alpha1 acid glycoprotein by using on line immunoextraction and high performance affinity chromatography
Journal of Chromatography A, 2017Co-Authors: Ryan Matsuda, William Clarke, Chenhua Zhang, Zitha Isingizwe, David S. HageAbstract:Abstract A method that combined on-line immunoextraction with high-performance affinity chromatography was developed to examine the binding of drugs with α 1 -acid glycoprotein (AGP). Affinity Microcolumns containing immobilized polyclonal anti-AGP antibodies were developed that had a capture efficiency of up to 98.4% for AGP and a binding capacity of 0.72 nmol AGP when using a 20 mm × 2.1 mm i.d. microcolumn. These Microcolumns were employed in various formats to examine the binding of drugs to normal AGP and AGP that had been adsorbed from serum samples for patients with systemic lupus erythematosus (SLE). Drugs that were screened in zonal elution experiments for their overall binding to these types of AGP included chlorpromazine, disopyramide, imipramine, propranolol, and warfarin. Most of these drugs showed an increase in their binding to the AGP from SLE serum when compared to normal AGP (i.e., an increase of 13–76%); however, disopyramide gave a 21–25% decrease in retention when the same AGP samples were compared. Frontal analysis was used to further evaluate the binding of disopyramide and imipramine to these forms of AGP. Both drugs gave a good fit to a model that involved a combination of saturable and non-saturable interactions with AGP. Changes in the non-saturable interactions accounted for most of variations seen in the binding of disopyramide and imipramine with the AGP samples. The methods used in this study could be adapted for use in personalized medicine and the study of other proteins or drugs using aqueous mixtures or clinical samples.
-
analysis of drug protein binding using on line immunoextraction and high performance affinity Microcolumns studies with normal and glycated human serum albumin
Journal of Chromatography A, 2015Co-Authors: Ryan Matsuda, Donald Jobe, Jared Beyersdorf, David S. HageAbstract:A method combining on-line immunoextraction Microcolumns with high-performance affinity chromatography (HPAC) was developed and tested for use in examining drug-protein interactions with normal or modified proteins. Normal human serum albumin (HSA) and glycated HSA were used as model proteins for this work. High-performance immunoextraction Microcolumns with sizes of 1.0-2.0 cm × 2.1mm i.d. and containing anti-HSA polyclonal antibodies were developed and tested for their ability to bind normal HSA or glycated HSA. These Microcolumns were able to extract up to 82-93% for either type of protein at 0.05-0.10 mL/min and had a binding capacity of 0.34-0.42 nmol HSA for a 1.0 cm × 2.1mm i.d. microcolumn. The immunoextraction Microcolumns and their adsorbed proteins were tested for use in various approaches for drug binding studies. Frontal analysis was used with the adsorbed HSA/glycated HSA to measure the overall affinities of these proteins for the drugs warfarin and gliclazide, giving comparable values to those obtained previously using similar protein preparations that had been covalently immobilized within HPAC columns. Zonal elution competition studies with gliclazide were next performed to examine the specific interactions of this drug at Sudlow sites I and II of the adsorbed proteins. These results were also comparable to those noted in prior work with covalently immobilized samples of normal HSA or glycated HSA. These experiments indicated that drug-protein binding studies can be carried out by using on-line immunoextraction Microcolumns with HPAC. The same method could be used in the future with clinical samples and other drugs or proteins of interest in pharmaceutical studies or biomedical research.
-
analysis of biomolecular interactions using affinity Microcolumns a review
Journal of Chromatography B, 2014Co-Authors: Xiwei Zheng, Erika L. Pfaunmiller, Na Tasha Carter, Ryan Matsuda, Sandya R Beeram, Maria Podariu, Christopher J White, David S. HageAbstract:Affinity chromatography has become an important tool for characterizing biomolecular interactions. The use of affinity Microcolumns, which contain immobilized binding agents and have volumes in the mid-to-low microliter range, has received particular attention in recent years. Potential advantages of affinity Microcolumns include the many analysis and detection formats that can be used with these columns, as well as the need for only small amounts of supports and immobilized binding agents. This review examines how affinity Microcolumns have been used to examine biomolecular interactions. Both capillary-based Microcolumns and short Microcolumns are considered. The use of affinity Microcolumns with zonal elution and frontal analysis methods are discussed. The techniques of peak decay analysis, ultrafast affinity extraction, split-peak analysis, and band-broadening studies are also explored. The principles of these methods are examined and various applications are provided to illustrate the use of these methods with affinity Microcolumns. It is shown how these techniques can be utilized to provide information on the binding strength and kinetics of an interaction, as well as on the number and types of binding sites. It is further demonstrated how information on competition or displacement effects can be obtained by these methods.
-
development of affinity Microcolumns for drug protein binding studies in personalized medicine interactions of sulfonylurea drugs with in vivo glycated human serum albumin
Analytical Chemistry, 2013Co-Authors: Jeanethe Anguizola, K S Joseph, Omar S Barnaby, Ryan Matsuda, Guadalupe Alvarado, William Clarke, Ronald L Cerny, David S. HageAbstract:This report used high-performance affinity Microcolumns to examine the changes in binding by sulfonylurea drugs to in vivo glycated HSA that had been isolated from individual patients with diabetes. An immunoextraction approach was developed to isolate HSA and glycated HSA from clinical samples, using only 20 μL of plasma or serum and 6–12 nmol of protein to prepare each affinity microcolumn. It was found that the affinity Microcolumns could be used in either frontal analysis or zonal elution studies, which typically required only 4–8 min per run. The Microcolumns had good stability and allowed data to be obtained for multiple drugs and experimental conditions over hundreds of sample application cycles. Both the overall binding, as measured by frontal analysis, and site-specific interactions, as examined by zonal elution, showed good agreement with previous data that had been obtained for in vitro glycated HSA with similar levels of modification. It was also possible to directly compare the changes in sit...
Juri Rappsilber - One of the best experts on this subject based on the ideXlab platform.
-
stop and go extraction tips for matrix assisted laser desorption ionization nanoelectrospray and lc ms sample pretreatment in proteomics
Analytical Chemistry, 2003Co-Authors: Juri Rappsilber, Yasushi Ishihama, Matthias MannAbstract:Proteomics is critically dependent on optimal sample preparation. Particularly, the interface between protein digestion and mass spectrometric analysis has a large influence on the overall quality and sensitivity of the analysis. We here describe a novel procedure in which a very small disk of beads embedded in a Teflon meshwork is placed as a microcolumn into pipet tips. Termed Stage, for STop And Go Extraction, the procedure has been implemented with commercially available material (C18 Empore Disks (3M, Minneapolis, MN)) as frit and separation material. The disk is introduced in a simple and fast process yielding a convenient and completely reliable procedure for the production of self-packed Microcolumns in pipet tips. It is held in place free of obstacles solely by the narrowing tip, ensuring optimized loading and elution of analytes. Five disks are conveniently placed in 1 min, adding 300 μL/...
-
stop and go extraction tips for matrix assisted laser desorption ionization nanoelectrospray and lc ms sample pretreatment in proteomics
Analytical Chemistry, 2003Co-Authors: Juri Rappsilber, Yasushi Ishihama, Matthias MannAbstract:Proteomics is critically dependent on optimal sample preparation. Particularly, the interface between protein digestion and mass spectrometric analysis has a large influence on the overall quality and sensitivity of the analysis. We here describe a novel procedure in which a very small disk of beads embedded in a Teflon meshwork is placed as a microcolumn into pipet tips. Termed Stage, for STop And Go Extraction, the procedure has been implemented with commercially available material (C18 Empore Disks (3M, Minneapolis, MN)) as frit and separation material. The disk is introduced in a simple and fast process yielding a convenient and completely reliable procedure for the production of self-packed Microcolumns in pipet tips. It is held in place free of obstacles solely by the narrowing tip, ensuring optimized loading and elution of analytes. Five disks are conveniently placed in 1 min, adding 300 μL/...
-
Microcolumns with self assembled particle frits for proteomics
Journal of Chromatography A, 2002Co-Authors: Yasushi Ishihama, Juri Rappsilber, Jens S Andersen, Matthias MannAbstract:LC-MS-MS experiments in proteomics are usually performed with packed Microcolumns employing frits or outlets smaller than the particle diameter to retain the packing material. We have developed packed Microcolumns using self-assembled particles (SAPs) as frits that are smaller than the size of the outlet. A five to one ratio of outlet size to particle diameter appears to be the upper maximum. In these situations the particles assembled into an arch over the outlet like the stones in a stone bridge. When 3 microm particles were packed into a tapered column with an 8 microm outlet, two particles bridged the outlet with 0.3 pl dead volume and perfect success rate. In peptide analysis by LC-MS, the peak width at half height was normally less than 6 s, compared to 12 s without SAPs. The LC-MS-MS system provided 37% sequence coverage (21 matched peptides) for a tryptically-digested sample of 10 fmol bovine serum albumin. We also describe application of the SAP principle to make disposable pipette tip columns with short pieces of fused-silica capillary as the outlet.
Martin R. Larsen - One of the best experts on this subject based on the ideXlab platform.
-
preparation of immobilized enzyme Microcolumns
CSH Protocols, 2007Co-Authors: Martin R. LarsenAbstract:INTRODUCTIONPeptide mapping by MALDI-TOF-MS has emerged as a powerful proteomics tool for identifying and characterizing proteins. One of the key steps in this method involves the proteolytic cleavage of proteins, followed by MS analysis of the generated peptides. A limitation of the method, especially for high-throughput proteomics, is the speed and efficiency of proteolytic cleavage. In this protocol, a method for rapid tryptic digestion using immobilized enzyme Microcolumns is described.
-
preparation of reversed phase Microcolumns
CSH Protocols, 2007Co-Authors: Martin R. LarsenAbstract:INTRODUCTIONOne versatile strategy for sample cleanup prior to MALDI-MS analysis uses microscale columns designed for direct sample elution onto the MALDI target plate. This protocol describes the fabrication of a reversed-phase microcolumn designed for this purpose. The Microcolumns are prepared from GELoader tips. This protocol has been optimized for sample cleanup prior to MALDI-MS. However, with slight modifications, it works equally well with samples destined for ESI-MS.
-
graphite powder as an alternative or supplement to reversed phase material for desalting and concentration of peptide mixtures prior to matrix assisted laser desorption ionization mass spectrometry
Proteomics, 2002Co-Authors: Martin R. Larsen, Stuart J Cordwell, Peter RoepstorffAbstract:The success attributed to identification and characterization of gel separated proteins by mass spectrometry (MS) is highly dependent on the percentage of an entire sequence covered by matching peptides derived from enzymatic digestion. Desalting and concentration of peptide mixtures on reversed-phase (RP) Microcolumns prior to mass spectrometric analysis have resulted in increased signal-to-noise ratio and sensitivity, and consequently higher sequence coverage. A large proportion of peptides, however, remains undetected by MS presumably because they are lost during sample preparation on Microcolumns, or are suppressed in the ionization process. We report here the use of graphite powder packed in constricted GELoader tips as an alternative to RP Microcolumns for desalting and concentration of peptide mixtures prior to MS. Such columns are able to retain small and/or hydrophilic peptides that can be lost when using RP Microcolumns. In addition, we show that samples contaminated with small biological polymers can readily be analyzed using graphite powder rather than RP Microcolumns, since the polymer molecules bind strongly to graphite and are not eluted with the peptides.