This document summarizes a study that evaluated a glycopeptide esterification method to better estimate sialylation levels and differentiate sialic acid linkages in monoclonal antibodies by MALDI-TOF mass spectrometry. Glycopeptides isolated from tryptic digests of monoclonal antibodies were ethyl esterified. Esterification neutralized sialic acids and decreased metastable fragmentation, allowing more accurate quantification of sialylation. Esterification patterns also enabled differentiation of α2,3 and α2,6 sialic acid linkages. The method was demonstrated to be fast, simple, and effective for monoclonal antibody glycopeptide analysis.
1) The document describes a novel method called INLIGHTTM for the relative quantification of N-linked glycans using isotopically labeled glycan hydrazide tags.
2) The method involves releasing N-linked glycans from glycoproteins using PNGase F, then derivatizing the glycans from two samples with either a light or heavy tagged reagent.
3) The tagged glycans are mixed, analyzed by LC/MS, and ratios of light and heavy glycan pairs are calculated to quantify differences between the two samples after correcting for isotopic overlap.
Biomimetic Glycoside Hydrolysis by a Microgel Templated with a Competitive Gl...aaaa zzzz
This document summarizes the synthesis and characterization of biomimetic microgel catalysts templated with a competitive glycosidase inhibitor (galactonoamidine). The microgels were synthesized via free radical polymerization in the presence of a binuclear copper complex and the inhibitor. Computational modeling was used to determine the inhibitor binds to the copper complex through its amidine and hydroxyl groups. The resulting microgels were characterized and found to have a narrow size distribution and near-quantitative incorporation of the copper complex. The microgels show potential as biomimetic catalysts for glycoside hydrolysis.
The document summarizes a study that analyzed 4 genetically engineered strains of Clostridium thermocellum with differing pathways for converting phosphoenolpyruvate (PEP) to pyruvate. The strains were cultured and their metabolite concentrations and reversibility of reactions were measured to calculate free energy changes. Strain 1138 diverted flux through the malate shunt, strain 1163 expressed an exogenous pyruvate kinase, and strain 1251 expressed pyruvate kinase and deleted PPDK and the malate shunt. Strain 1163 had higher GDP and GTP, suggesting its PEP to oxaloacetate conversion was more efficient. Further optimizing the PEP to pyruvate pathway, such as
Gelatin-grafted N- proflavine acryl amide was synthesized through two steps; firstly the Gelatin was grafted with
acrylic acid free radically using Ammonium per-sulfate at 60℃, Then it was modified to its corresponding acyl
chloride derivation, second step included the substitution with amino group of proflavine, in this research Gelatin
was used as a natural nontoxic, water soluble polymer as a drug carrier.
The prepared pro drug polymer was characterized by FTIR and 1H-NMR spectroscopies, Controlled drug release
was studied in different pH values at 37℃. Many advantages were obtained comparing with other known
methods.
This document describes research into developing new inhibitors that can selectively target specific calpain isoforms for therapeutic applications. The researcher synthesized a potential calpain inhibitor incorporating a symmetrical (1-2-dithiolan-4-yl) carboxylic acid into a dipeptide motif. Coupling reactions using propylphosphonic anhydride generally worked well but coupling 2-bromo-acrylic acid was complicated. Future work involves further characterizing products, synthesizing the dithiolane acid, and evaluating inhibitors against calpain enzymes.
solid phase synthesis Presentation by komalKomal Rajgire
The document summarizes solid phase synthesis. It begins with an introduction describing how solid phase synthesis involves coupling reagents to a solid support to perform multi-step reactions leading to a target molecule. It then discusses various aspects of planning solid phase synthesis such as suitable resin supports, linkers, protective groups, and monitoring reactions. Examples of resin types, linkers, and protective groups are provided. The document concludes by outlining advantages such as simplified purification and green chemistry principles, as well as disadvantages such as potential low reaction rates. Applications mentioned include combinatorial synthesis, peptide synthesis, and DNA synthesis.
This document describes research on the synthesis of anti-1,2-diols using aldehyde α-oxygenation followed by organometallic addition. Contrary to previous reports, the researchers found this method produces anti-diols, not syn-diols. They demonstrated the scope of the reaction using different organometallic reagents and applied it to synthesize stereoisomers of oxylipins from a plant, determining the stereochemistry of two natural products. The synthesis involved aldehyde α-oxygenation, organometallic addition, functional group manipulations and comparisons to natural products to assign stereochemistry.
1) The document describes a novel method called INLIGHTTM for the relative quantification of N-linked glycans using isotopically labeled glycan hydrazide tags.
2) The method involves releasing N-linked glycans from glycoproteins using PNGase F, then derivatizing the glycans from two samples with either a light or heavy tagged reagent.
3) The tagged glycans are mixed, analyzed by LC/MS, and ratios of light and heavy glycan pairs are calculated to quantify differences between the two samples after correcting for isotopic overlap.
Biomimetic Glycoside Hydrolysis by a Microgel Templated with a Competitive Gl...aaaa zzzz
This document summarizes the synthesis and characterization of biomimetic microgel catalysts templated with a competitive glycosidase inhibitor (galactonoamidine). The microgels were synthesized via free radical polymerization in the presence of a binuclear copper complex and the inhibitor. Computational modeling was used to determine the inhibitor binds to the copper complex through its amidine and hydroxyl groups. The resulting microgels were characterized and found to have a narrow size distribution and near-quantitative incorporation of the copper complex. The microgels show potential as biomimetic catalysts for glycoside hydrolysis.
The document summarizes a study that analyzed 4 genetically engineered strains of Clostridium thermocellum with differing pathways for converting phosphoenolpyruvate (PEP) to pyruvate. The strains were cultured and their metabolite concentrations and reversibility of reactions were measured to calculate free energy changes. Strain 1138 diverted flux through the malate shunt, strain 1163 expressed an exogenous pyruvate kinase, and strain 1251 expressed pyruvate kinase and deleted PPDK and the malate shunt. Strain 1163 had higher GDP and GTP, suggesting its PEP to oxaloacetate conversion was more efficient. Further optimizing the PEP to pyruvate pathway, such as
Gelatin-grafted N- proflavine acryl amide was synthesized through two steps; firstly the Gelatin was grafted with
acrylic acid free radically using Ammonium per-sulfate at 60℃, Then it was modified to its corresponding acyl
chloride derivation, second step included the substitution with amino group of proflavine, in this research Gelatin
was used as a natural nontoxic, water soluble polymer as a drug carrier.
The prepared pro drug polymer was characterized by FTIR and 1H-NMR spectroscopies, Controlled drug release
was studied in different pH values at 37℃. Many advantages were obtained comparing with other known
methods.
This document describes research into developing new inhibitors that can selectively target specific calpain isoforms for therapeutic applications. The researcher synthesized a potential calpain inhibitor incorporating a symmetrical (1-2-dithiolan-4-yl) carboxylic acid into a dipeptide motif. Coupling reactions using propylphosphonic anhydride generally worked well but coupling 2-bromo-acrylic acid was complicated. Future work involves further characterizing products, synthesizing the dithiolane acid, and evaluating inhibitors against calpain enzymes.
solid phase synthesis Presentation by komalKomal Rajgire
The document summarizes solid phase synthesis. It begins with an introduction describing how solid phase synthesis involves coupling reagents to a solid support to perform multi-step reactions leading to a target molecule. It then discusses various aspects of planning solid phase synthesis such as suitable resin supports, linkers, protective groups, and monitoring reactions. Examples of resin types, linkers, and protective groups are provided. The document concludes by outlining advantages such as simplified purification and green chemistry principles, as well as disadvantages such as potential low reaction rates. Applications mentioned include combinatorial synthesis, peptide synthesis, and DNA synthesis.
This document describes research on the synthesis of anti-1,2-diols using aldehyde α-oxygenation followed by organometallic addition. Contrary to previous reports, the researchers found this method produces anti-diols, not syn-diols. They demonstrated the scope of the reaction using different organometallic reagents and applied it to synthesize stereoisomers of oxylipins from a plant, determining the stereochemistry of two natural products. The synthesis involved aldehyde α-oxygenation, organometallic addition, functional group manipulations and comparisons to natural products to assign stereochemistry.
Synthesis, characterization, amdet and docking studies of novel diclofenac de...Alexander Decker
This document discusses the synthesis and characterization of novel diclofenac derivatives containing phenylalanine moieties as selective inhibitors of cyclooxygenase-2 (COX-2). Sixteen novel diclofenac derivatives were synthesized and their structures were confirmed through various analytical techniques. Molecular docking studies predicted that compounds 7, 12 and 16 showed stronger binding with COX-2 than the reference drug diclofenac, making them potential selective COX-2 inhibitors. The binding scores of these compounds were higher than diclofenac when docked into the active site of COX-2.
plain 4 SYNTHESIS, PURIFICATION AND STABILITY STUDY OF ISLETAndrew Apals
This document outlines Andrew Apals' thesis defense presentation on the synthesis, purification, and stability study of the islet neogenesis-associated protein peptide (INGAP-P) and analogs. The presentation covers solid phase peptide synthesis of INGAP-P, linear and cyclic analogs. It also discusses reverse-phase HPLC method development for the separation and quantification of peptides in synthetic mixtures, including degradation studies of INGAP-P standards. The goal of the research is to develop purification methods to resolve INGAP-P from crude synthesis mixtures using analytical HPLC instrumentation.
This document describes research into developing a single chemical entity that can deliver two antimalarial drugs through independent mechanisms of action. Specifically, it details the synthesis of endoperoxide-carbonyl inhibitor hybrid molecules designed to target the malaria parasite. Upon decomposition by heme or ferrous iron in the parasite, these hybrids are intended to simultaneously release a potentially cytotoxic carbon-centered radical as well as a cysteine protease inhibitor, targeting the parasite through two pathways and reducing the risk of resistance development. The researchers synthesized both carbonyl-containing peptide inhibitors and 1,2,4-trioxolane prodrugs, finding that the aldehyde and ketone versions showed nanomolar inhibitory activity against falcipain cysteine proteases
1. The study examines how the substrate L-serine interacts with and stabilizes the active site of serine hydroxymethyltransferase (SHMT) enzymes from sheep liver and E. coli.
2. Results show L-serine enhances the thermal stability of both SHMT enzymes, increasing their apparent melting temperatures. Binding studies also indicate L-serine induces conformational changes in the enzymes without altering their overall structure.
3. The conformational changes upon L-serine binding, monitored by various spectroscopic methods, suggest L-serine compacts the enzyme structure, leading to increased thermal stability.
Synthesis & analysis of surfactant chelating precursors onSUMMAH aswin
The document discusses surfactant chemistry and properties. Surfactants are amphiphilic molecules with both hydrophobic and hydrophilic groups that arrange themselves at interfaces. They can form micelles in solution, with hydrophobic regions shielded within spherical structures. The critical micelle concentration depends on factors like chain length and temperature. Different techniques are used to determine the stoichiometric ratios and binding constants of metal-ligand complexes like copper complexes. Ratios and binding strengths are found to vary with technique and conditions.
The document discusses methodology in organic synthesis, including examples of natural products. It describes convergent and divergent synthesis strategies. Convergent synthesis involves coupling molecular fragments through independent synthesis to improve reaction yields compared to linear synthesis. Divergent synthesis starts from a central core and generates a library of compounds through successive additions. Functional group interconversion and addition techniques are discussed to allow for disconnection of target molecules during retrosynthetic analysis.
This study investigated the effects of substituting the native haem group in iNOS with mesohaem, which has a higher electron density. The goals were to dissect the structural and electronic effects on iNOS catalysis. Key findings include:
1) iNOS and the W188H mutant with mesohaem substitution were stable and dimeric, and had similar substrate binding affinities as their native haem counterparts.
2) Single turnover kinetic experiments showed mesohaem substitution triggered higher rates of dioxygen conversion and altered other kinetic parameters.
3) The first crystal structure of iNOS with mesohaem substitution showed essentially identical features to native iNOS, indicating electronic effects primarily influence kinetics rather
Dynamic modification of PMMA chips using PVA for GAG disaccharide isomer sepa...Yong Zhang
This document describes a microchip electrophoresis (MCE) method for separating unsaturated disaccharides from glycosaminoglycans (GAGs) using poly(methyl methacrylate) (PMMA) microchips dynamically coated with poly(vinyl alcohol) (PVA). PVA coating was shown to increase the hydrophilicity of the PMMA surface and reduce nonspecific adsorption. Using PVA-coated PMMA chips, two pairs of GAG disaccharide isomers (nDi-diSB/nDi-diSD and nDi-0S/nDi-HA) were baseline separated within 130 seconds by MCE for the first time. The dynamic PVA coating approach improves MCE resolution for
This document summarizes a study that investigated the effects of two disaccharides (trehalose and sucrose) and trimethylamine N-oxide (TMAO) on amyloid-beta (Aβ) aggregation and interaction with lipid membranes. The key findings were:
1) In the absence of lipid vesicles, trehalose and sucrose delayed Aβ aggregation as measured by Thioflavin T fluorescence, but TMAO did not affect aggregation.
2) In the presence of lipid vesicles, all three osmolytes (trehalose, sucrose, TMAO) significantly attenuated dye leakage from the vesicles induced by Aβ aggregates.
3) Hydrogen exchange mass spectrometry (HX-MS) and
This document describes the design, synthesis, and evaluation of a series of 1,3-disubstituted pyrrolo[2,3-b]quinoxalines as potential inhibitors of phosphodiesterase 4 (PDE4) and cancer cell growth. A ligand- and phase transfer catalyst-free intramolecular Heck reaction was used to synthesize the target compounds. Some compounds showed significant inhibition of PDE4B and growth inhibition of oral cancer cells in vitro. They also showed acceptable safety profiles in zebrafish embryos, but no apoptosis was observed. The goal was to develop PDE4 inhibitors that do not inhibit luciferase, which could produce false positives in assays.
This document summarizes research exploring the structure and function of HMG-CoA reductase (HMGR) in Burkholderia cenocepacia. Key findings include:
1) B. cenocepacia HMGR (BcHMGR) exhibits properties of a morpheein, meaning it can exist in multiple quaternary structure states that influence enzymatic activity.
2) The equilibrium between BcHMGR structure states is affected by factors like ligand concentration, enzyme concentration, and pH.
3) BcHMGR preferentially catalyzes the oxidation of mevalonate to HMG-CoA, unlike most HMGRs which catalyze the reverse reaction.
The document discusses retrosynthetic analysis, which is a problem-solving technique used in organic synthesis. It involves working backwards from the target molecule and breaking it down into simpler structures through the reverse of known reactions. This allows chemists to plan a synthesis by tracing the target back to commercially available starting materials. The document outlines common disconnections, reactions, and strategies used in retrosynthetic analysis and organic synthesis planning.
Enhanced electrophoretic resolution of monosulfate GAG disacchride isomers on...Yong Zhang
This document describes research on improving the separation of monosulfate glycosaminoglycan disaccharide isomers by microchip electrophoresis. Key findings include:
1) Addition of 1,4-dioxane (DO) to the running buffer dramatically improved resolution of the isomers, likely due to solvation effects.
2) Methylcellulose was used to suppress electroosmotic flow and analyte adsorption to the poly(methyl methacrylate) microchip surface.
3) Optimization of buffer pH, addition of beta-cyclodextrin, and concentration of 1,4-dioxane enhanced resolution of the monosulfate isomers.
4) Under optimized conditions,
1) Researchers used fragment-based crystallography screening and medicinal chemistry to develop inhibitors of leukotriene A4 hydrolase (LTA4H), identifying compound 20 (DG-051) as a potent inhibitor.
2) They identified initial fragment hits that bound to LTA4H's active site through crystallography screening and optimized fragments through iterative chemistry and structural analysis, guided by ligand efficiency.
3) Compound 20 (DG-051) was identified as a potent, orally bioavailable inhibitor of LTA4H currently in clinical trials for myocardial infarction and stroke.
1. The document describes the design and synthesis of molecules that mimic the natural selectin ligand sialyl Lewis X (sLeX) to function as selectin antagonists.
2. Key modifications to the sLeX structure included replacing the GlcNAc saccharide unit with an acyclic tether, and installing benzoate groups at different positions on the galactose and fucose units.
3. The new molecules were evaluated for their ability to inhibit selectin interactions using assays with E-selectin and P-selectin. Compounds with benzoate groups at both C2 and C4 positions on galactose showed the highest potency as selectin antagonists.
This document summarizes a study on the interaction of pyridoxal-5'-phosphate (PLP) with the apo form of sheep liver serine hydroxymethyltransferase (SHMT). The key findings are:
1) Removal of PLP from the holoenzyme converts it to the inactive apoenzyme and addition of PLP back restores full enzyme activity, demonstrating PLP's role in catalysis.
2) PLP binding to the apoenzyme occurs in two phases, a very rapid initial phase and a slower secondary phase, forming an internal aldimine linkage critical for activity.
3) While the secondary structures of the apo and holo forms are identical, they have different
This document describes a study that synthesized a new class of tyrosinase inhibitors called azachalcones. Azachalcone derivatives were tested for their ability to inhibit the enzyme tyrosinase, which is involved in melanin biosynthesis. Two compounds that were reduction products of pyridinyl azachalcones strongly inhibited tyrosinase activity and were more potent inhibitors than the positive control kojic acid. Kinetic studies showed that these two compounds act as competitive inhibitors of tyrosinase by binding to the enzyme's active site. This new class of azachalcone inhibitors could potentially be used as depigmenting agents or to prevent browning in foods.
This document summarizes research on the effects of heme ring oxygenation on the structure and function of cytochrome c peroxidase (CcP). Specifically, it describes the synthesis of 4-mesoporphyrinone (mesopone) and its incorporation into CcP to form a hybrid protein called MpCcP. Testing found that MpCcP had similar peroxidase activity to wild-type CcP with cytochrome c, but varied activity with other substrates. Structural analysis via X-ray crystallography provided the first structural characterization of an oxygenated heme protein and found only the S-isomer of mesopone in the crystallized protein despite using a mixture of isomers.
1) Nucleotides and phosphorylation can both positively and negatively modulate the binding of CaMKII to the NMDA receptor subunit GluN2B.
2) Adding ATP to in vitro experiments enhanced CaMKII binding to GluN2B, through two positive effects (direct nucleotide binding and CaMKII autophosphorylation) and two negative effects (GluN2B phosphorylation and additional CaMKII autophosphorylation).
3) Within cells, where ATP levels are high, nucleotide binding was required for efficient CaMKII interaction with GluN2B, whereas CaMKII autophosphorylation was not, suggesting nucleotide binding acts faster than the inhibitory phosphorylation reactions.
This document describes a study that used solid-phase microextraction (SPME) combined with gas chromatography-mass spectrometry (GC-MS) to characterize metabolites produced during the biodesulfurization of two model organosulfur compounds, dibenzothiophene (DBT) and 4,6-diethyl-dibenzothiophene (DEDBT), by Rhodococcus sp. strain ECRD-1. The following metabolites were identified for DBT: DBT sulfoxide, DBT sulfone, dibenz[c,e][1,2]oxathiin 6-oxide (sultine), dibenz[c,e][1,2]oxathiin
Synthesis, characterization, amdet and docking studies of novel diclofenac de...Alexander Decker
This document discusses the synthesis and characterization of novel diclofenac derivatives containing phenylalanine moieties as selective inhibitors of cyclooxygenase-2 (COX-2). Sixteen novel diclofenac derivatives were synthesized and their structures were confirmed through various analytical techniques. Molecular docking studies predicted that compounds 7, 12 and 16 showed stronger binding with COX-2 than the reference drug diclofenac, making them potential selective COX-2 inhibitors. The binding scores of these compounds were higher than diclofenac when docked into the active site of COX-2.
plain 4 SYNTHESIS, PURIFICATION AND STABILITY STUDY OF ISLETAndrew Apals
This document outlines Andrew Apals' thesis defense presentation on the synthesis, purification, and stability study of the islet neogenesis-associated protein peptide (INGAP-P) and analogs. The presentation covers solid phase peptide synthesis of INGAP-P, linear and cyclic analogs. It also discusses reverse-phase HPLC method development for the separation and quantification of peptides in synthetic mixtures, including degradation studies of INGAP-P standards. The goal of the research is to develop purification methods to resolve INGAP-P from crude synthesis mixtures using analytical HPLC instrumentation.
This document describes research into developing a single chemical entity that can deliver two antimalarial drugs through independent mechanisms of action. Specifically, it details the synthesis of endoperoxide-carbonyl inhibitor hybrid molecules designed to target the malaria parasite. Upon decomposition by heme or ferrous iron in the parasite, these hybrids are intended to simultaneously release a potentially cytotoxic carbon-centered radical as well as a cysteine protease inhibitor, targeting the parasite through two pathways and reducing the risk of resistance development. The researchers synthesized both carbonyl-containing peptide inhibitors and 1,2,4-trioxolane prodrugs, finding that the aldehyde and ketone versions showed nanomolar inhibitory activity against falcipain cysteine proteases
1. The study examines how the substrate L-serine interacts with and stabilizes the active site of serine hydroxymethyltransferase (SHMT) enzymes from sheep liver and E. coli.
2. Results show L-serine enhances the thermal stability of both SHMT enzymes, increasing their apparent melting temperatures. Binding studies also indicate L-serine induces conformational changes in the enzymes without altering their overall structure.
3. The conformational changes upon L-serine binding, monitored by various spectroscopic methods, suggest L-serine compacts the enzyme structure, leading to increased thermal stability.
Synthesis & analysis of surfactant chelating precursors onSUMMAH aswin
The document discusses surfactant chemistry and properties. Surfactants are amphiphilic molecules with both hydrophobic and hydrophilic groups that arrange themselves at interfaces. They can form micelles in solution, with hydrophobic regions shielded within spherical structures. The critical micelle concentration depends on factors like chain length and temperature. Different techniques are used to determine the stoichiometric ratios and binding constants of metal-ligand complexes like copper complexes. Ratios and binding strengths are found to vary with technique and conditions.
The document discusses methodology in organic synthesis, including examples of natural products. It describes convergent and divergent synthesis strategies. Convergent synthesis involves coupling molecular fragments through independent synthesis to improve reaction yields compared to linear synthesis. Divergent synthesis starts from a central core and generates a library of compounds through successive additions. Functional group interconversion and addition techniques are discussed to allow for disconnection of target molecules during retrosynthetic analysis.
This study investigated the effects of substituting the native haem group in iNOS with mesohaem, which has a higher electron density. The goals were to dissect the structural and electronic effects on iNOS catalysis. Key findings include:
1) iNOS and the W188H mutant with mesohaem substitution were stable and dimeric, and had similar substrate binding affinities as their native haem counterparts.
2) Single turnover kinetic experiments showed mesohaem substitution triggered higher rates of dioxygen conversion and altered other kinetic parameters.
3) The first crystal structure of iNOS with mesohaem substitution showed essentially identical features to native iNOS, indicating electronic effects primarily influence kinetics rather
Dynamic modification of PMMA chips using PVA for GAG disaccharide isomer sepa...Yong Zhang
This document describes a microchip electrophoresis (MCE) method for separating unsaturated disaccharides from glycosaminoglycans (GAGs) using poly(methyl methacrylate) (PMMA) microchips dynamically coated with poly(vinyl alcohol) (PVA). PVA coating was shown to increase the hydrophilicity of the PMMA surface and reduce nonspecific adsorption. Using PVA-coated PMMA chips, two pairs of GAG disaccharide isomers (nDi-diSB/nDi-diSD and nDi-0S/nDi-HA) were baseline separated within 130 seconds by MCE for the first time. The dynamic PVA coating approach improves MCE resolution for
This document summarizes a study that investigated the effects of two disaccharides (trehalose and sucrose) and trimethylamine N-oxide (TMAO) on amyloid-beta (Aβ) aggregation and interaction with lipid membranes. The key findings were:
1) In the absence of lipid vesicles, trehalose and sucrose delayed Aβ aggregation as measured by Thioflavin T fluorescence, but TMAO did not affect aggregation.
2) In the presence of lipid vesicles, all three osmolytes (trehalose, sucrose, TMAO) significantly attenuated dye leakage from the vesicles induced by Aβ aggregates.
3) Hydrogen exchange mass spectrometry (HX-MS) and
This document describes the design, synthesis, and evaluation of a series of 1,3-disubstituted pyrrolo[2,3-b]quinoxalines as potential inhibitors of phosphodiesterase 4 (PDE4) and cancer cell growth. A ligand- and phase transfer catalyst-free intramolecular Heck reaction was used to synthesize the target compounds. Some compounds showed significant inhibition of PDE4B and growth inhibition of oral cancer cells in vitro. They also showed acceptable safety profiles in zebrafish embryos, but no apoptosis was observed. The goal was to develop PDE4 inhibitors that do not inhibit luciferase, which could produce false positives in assays.
This document summarizes research exploring the structure and function of HMG-CoA reductase (HMGR) in Burkholderia cenocepacia. Key findings include:
1) B. cenocepacia HMGR (BcHMGR) exhibits properties of a morpheein, meaning it can exist in multiple quaternary structure states that influence enzymatic activity.
2) The equilibrium between BcHMGR structure states is affected by factors like ligand concentration, enzyme concentration, and pH.
3) BcHMGR preferentially catalyzes the oxidation of mevalonate to HMG-CoA, unlike most HMGRs which catalyze the reverse reaction.
The document discusses retrosynthetic analysis, which is a problem-solving technique used in organic synthesis. It involves working backwards from the target molecule and breaking it down into simpler structures through the reverse of known reactions. This allows chemists to plan a synthesis by tracing the target back to commercially available starting materials. The document outlines common disconnections, reactions, and strategies used in retrosynthetic analysis and organic synthesis planning.
Enhanced electrophoretic resolution of monosulfate GAG disacchride isomers on...Yong Zhang
This document describes research on improving the separation of monosulfate glycosaminoglycan disaccharide isomers by microchip electrophoresis. Key findings include:
1) Addition of 1,4-dioxane (DO) to the running buffer dramatically improved resolution of the isomers, likely due to solvation effects.
2) Methylcellulose was used to suppress electroosmotic flow and analyte adsorption to the poly(methyl methacrylate) microchip surface.
3) Optimization of buffer pH, addition of beta-cyclodextrin, and concentration of 1,4-dioxane enhanced resolution of the monosulfate isomers.
4) Under optimized conditions,
1) Researchers used fragment-based crystallography screening and medicinal chemistry to develop inhibitors of leukotriene A4 hydrolase (LTA4H), identifying compound 20 (DG-051) as a potent inhibitor.
2) They identified initial fragment hits that bound to LTA4H's active site through crystallography screening and optimized fragments through iterative chemistry and structural analysis, guided by ligand efficiency.
3) Compound 20 (DG-051) was identified as a potent, orally bioavailable inhibitor of LTA4H currently in clinical trials for myocardial infarction and stroke.
1. The document describes the design and synthesis of molecules that mimic the natural selectin ligand sialyl Lewis X (sLeX) to function as selectin antagonists.
2. Key modifications to the sLeX structure included replacing the GlcNAc saccharide unit with an acyclic tether, and installing benzoate groups at different positions on the galactose and fucose units.
3. The new molecules were evaluated for their ability to inhibit selectin interactions using assays with E-selectin and P-selectin. Compounds with benzoate groups at both C2 and C4 positions on galactose showed the highest potency as selectin antagonists.
This document summarizes a study on the interaction of pyridoxal-5'-phosphate (PLP) with the apo form of sheep liver serine hydroxymethyltransferase (SHMT). The key findings are:
1) Removal of PLP from the holoenzyme converts it to the inactive apoenzyme and addition of PLP back restores full enzyme activity, demonstrating PLP's role in catalysis.
2) PLP binding to the apoenzyme occurs in two phases, a very rapid initial phase and a slower secondary phase, forming an internal aldimine linkage critical for activity.
3) While the secondary structures of the apo and holo forms are identical, they have different
This document describes a study that synthesized a new class of tyrosinase inhibitors called azachalcones. Azachalcone derivatives were tested for their ability to inhibit the enzyme tyrosinase, which is involved in melanin biosynthesis. Two compounds that were reduction products of pyridinyl azachalcones strongly inhibited tyrosinase activity and were more potent inhibitors than the positive control kojic acid. Kinetic studies showed that these two compounds act as competitive inhibitors of tyrosinase by binding to the enzyme's active site. This new class of azachalcone inhibitors could potentially be used as depigmenting agents or to prevent browning in foods.
This document summarizes research on the effects of heme ring oxygenation on the structure and function of cytochrome c peroxidase (CcP). Specifically, it describes the synthesis of 4-mesoporphyrinone (mesopone) and its incorporation into CcP to form a hybrid protein called MpCcP. Testing found that MpCcP had similar peroxidase activity to wild-type CcP with cytochrome c, but varied activity with other substrates. Structural analysis via X-ray crystallography provided the first structural characterization of an oxygenated heme protein and found only the S-isomer of mesopone in the crystallized protein despite using a mixture of isomers.
1) Nucleotides and phosphorylation can both positively and negatively modulate the binding of CaMKII to the NMDA receptor subunit GluN2B.
2) Adding ATP to in vitro experiments enhanced CaMKII binding to GluN2B, through two positive effects (direct nucleotide binding and CaMKII autophosphorylation) and two negative effects (GluN2B phosphorylation and additional CaMKII autophosphorylation).
3) Within cells, where ATP levels are high, nucleotide binding was required for efficient CaMKII interaction with GluN2B, whereas CaMKII autophosphorylation was not, suggesting nucleotide binding acts faster than the inhibitory phosphorylation reactions.
This document describes a study that used solid-phase microextraction (SPME) combined with gas chromatography-mass spectrometry (GC-MS) to characterize metabolites produced during the biodesulfurization of two model organosulfur compounds, dibenzothiophene (DBT) and 4,6-diethyl-dibenzothiophene (DEDBT), by Rhodococcus sp. strain ECRD-1. The following metabolites were identified for DBT: DBT sulfoxide, DBT sulfone, dibenz[c,e][1,2]oxathiin 6-oxide (sultine), dibenz[c,e][1,2]oxathiin
Expression Purification and Immunodetection of a fusion protein Glutathione S...iosrjce
Glutathione S Transferase(GST) is an enzyme of a multi gene family which is involved in reducing
oxidative damage to cells and detoxification of Xenobiotic compounds and plays critical role in life processes.
The entire work was completely qualitative and the objective of my work was to deal with the induction,
extraction and purification of the GST fusion protein from pGEX 3X vector.In order to achieve high degree of
transformed cells,the E.Coli BL21 host strain was made competent using 0.1M CaCl2 and adding of pGEX 3X
vector into host made it transformed.With the induction of GST protein by 0.1mM IPTG,the desired protein was
purified through glutathione Cl agarose column and was detected by immunoblotting method with the use of
anti GST HRP conjugate Ab which expressed the desired protein.
Expression Purification and Immunodetection of a fusion protein Glutathione S...iosrjce
IOSR Journal of Biotechnology and Biochemistry (IOSR-JBB) covers studies of the chemical processes in living organisms, structure and function of cellular components such as proteins, carbohydrates, lipids, nucleic acids and other biomolecules, chemical properties of important biological molecules, like proteins, in particular the chemistry of enzyme-catalyzed reactions, genetic code (DNA, RNA), protein synthesis, cell membrane transport, and signal transduction. IOSR-JBB is privileged to focus on a wide range of biotechnology as well as high quality articles on genetic engineering, cell and tissue culture technologies, genetics, microbiology, molecular biology, biochemistry, embryology, cell biology, chemical engineering, bioprocess engineering, information technology, biorobotics.
This document summarizes a study comparing the metabolism of cancerous K562 leukemia cells to non-cancerous CD34+ cells. Assays were performed to measure the activity of lactose dehydrogenase (LDH), an enzyme involved in glycolysis, and cytochrome c oxidase, involved in oxidative phosphorylation. Results showed that LDH activity was over twice as high in K562 cells compared to CD34+ cells, indicating K562 cells prioritize glycolysis over oxidative phosphorylation for energy production. While cytochrome c oxidase assay results were inconclusive, the LDH findings support the Warburg hypothesis that cancer cells favor glycolysis even in non-hypoxic conditions.
This study examined the interaction between the Abl kinase domain and the cancer drug Gleevec (imatinib) using site-directed mutagenesis to introduce a mutation (S417Y) associated with drug resistance. Researchers used PCR, protein purification techniques, and kinase assays to compare the inhibitory effect of Gleevec on wild-type Abl and the S417Y mutant. Their results supported the hypothesis that the S417Y mutation decreases Gleevec's ability to inhibit Abl kinase activity, though protein impurities limited the strength of the conclusions. The findings help explain why some cancers become resistant to Gleevec treatment.
This document describes a method for enriching and identifying O-GlcNAc-modified proteins and sites using click chemistry, on-resin digestion, and selective β-elimination. The method involves metabolic labeling of cells with azide-modified GlcNAc, click chemistry to purify labeled proteins, on-resin digestion, and β-elimination to selectively tag and identify former O-GlcNAc sites. Using this approach, the authors identified over 1500 O-GlcNAc proteins and 185 modification sites from a single cell line, demonstrating the utility of the method for proteome-wide analysis of O-GlcNAcylation. They also applied the method to study the effects of an
Applications of recombinant DNA technology – Production of Secondary Metabolites Synthesis of commercial products: Amino acids, ascorbic acid and novel antibiotics.
The role of GSH protection from DGA’s toxicity using digitonin fractionation ...Sarah Lopez
1) The document reports on an experiment studying the metabolic pathway of diethylene glycol (DEG) and the role of glutathione (GSH) in protecting against toxicity.
2) The experiment used a digitonin fractionation technique to separate cell fractions and measured enzyme activity to confirm separation of cytosolic and mitochondrial components.
3) Preliminary results showed higher lactate dehydrogenase activity in the cytosolic fraction and higher ketoglutarate dehydrogenase activity in the mitochondrial pellet fraction, supporting successful separation. Further study of GSH levels is planned.
This study investigated the effects of antioxidants such as alpha-lipoic acid, vitamin C, and resveratrol on the overexpression of cytochrome P450 2E1 in streptozotocin-induced diabetic rat tissues. Real-time PCR and RT-PCR analysis showed that administration of vitamin C to diabetic rats had the most significant effect in reducing CYP2E1 overexpression, while administration of vitamin C together with alpha-lipoic acid caused the second most decrease. Administration of alpha-lipoic acid alone resulted in lower reduction of CYP2E1 expression compared to vitamin C and the combination of vitamins.
The document discusses the purification of a Sitag/RGD/His-tag fusion protein for use as a scaffold in tissue engineering. It examines different lysis and purification methods to extract the protein from E. coli cells. Lysing methods tested include freeze-thaw, detergents, and enzymes. Purification was initially attempted using nickel affinity chromatography via the His-tag, but residual proteins remained. Purification using silica and the silica-binding Sitag was then explored, but elution with L-lysine was ineffective. Increased washes and incubation improved elution, but some bacterial proteins still remained. Future work may use a silica gel system or alter purification conditions.
This document describes the purification and characterization of a trypanothione-glutathione thioltransferase enzyme from Trypanosoma cruzi. Key findings include:
1) A 52 kDa protein was purified from T. cruzi using affinity chromatography columns containing either S-hexylglutathione or a trypanothione disulfide analogue as ligands.
2) Partial amino acid sequencing showed this protein is identical to one encoded by the previously described TcAc2 cDNA.
3) While it does not have significant glutathione or trypanothione transferase activity, it was found to catalyze thiol-disulfide exchange between dihydrotrypanothione and glutathione dis
Post-translational modification of monoclonal antibodiesSOMAYEH BAKHSHI
This document discusses post-translational modification of monoclonal antibodies. It begins by defining monoclonal antibodies and describing their basic structure, which includes heavy and light chains joined by disulfide bonds. It then lists several types of common post-translational modifications for monoclonal antibodies, including glycosylation, deamidation, isomerization, oxidation, and variants involving cysteines. For each modification type, it provides brief details about the chemical process, affected amino acids, impact on structure and function, and factors that influence the rate of modification.
This study investigated how changes in cadherin structure at the cell surface regulate adhesive activity. The researchers found:
1) Activating monoclonal antibodies recognize conformational epitopes near calcium binding sites between cadherin domains, inducing adhesion.
2) Antibody activation induces dephosphorylation of specific residues in p120-catenin's N-terminal domain, mediating adhesion.
3) Phosphorylation-deficient and phosphomimetic p120 mutants in cells had opposite effects on adhesion, indicating p120 phosphorylation directly controls cadherin adhesion.
Determination of DNA Methylation Using Electrochemiluminescenc.docxkhenry4
Determination of DNA Methylation Using Electrochemiluminescence
with Surface Accumulable Coreactant
Ryoji Kurita,*,† Kumi Arai,† Kohei Nakamoto,†,‡ Dai Kato,† and Osamu Niwa†,‡
†National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki,
Japan 305-8566
‡Graduate School of Pure and Applied Science, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki, Japan 305-8573
ABSTRACT: Cytosine methylation in DNA was determined by
an enzyme linked immunosorbent assay (ELISA) with electro-
chemiluminescence (ECL) detection and employed for the DNA
methylation assay of a long and real genomic sample for the first
time. The developed method employed an antimethyl cytosine
antibody labeled with acetylcholinesterase, which was added to
recognize single methylated cytosine in a DNA oligomer. The
acetylcholinesterase converted acetylthiocholine (substrate) to
thiocholine (product), which was accumulated on a gold electrode
surface via gold−thiol binding. This surface accumulated
preconcentration made it possible to observe bright and distinctive
ECL by applying a potential to the gold electrode in the presence
of a tris(2,2-bipyridyl)ruthenium complex luminophore when the
analyte DNA contained a methylation region. Methyl-cytosine was measured quantitatively in the 1−100 pmol range, which
exhibits sufficiently high sensitivity to achieve real DNA measurements without amplification by a polymerase chain reaction
(PCR). The proposed ECL method also exhibited high selectivity for methyl-cytosine against nonmethylated cytosine, guanine,
thymine, and adenine nucleotides. Finally, original and methylated DNA samples were clearly distinguished with our method
using a real DNA bacteriophage sample (48 502 base pairs).
DNA methylation is a well-known epigenetic modificationmechanism that regulates gene expression and plays
crucial roles in embryonic development.1 Cytosine methylation
in CpG islands has received particular attention because it is
thought to be involved in controlling genetic expression,
including that in cancer,2 genomic imprinting,3 cellular
differentiation, and Alzheimer’s disease.4 5-Methyl-cytosine is
now recognized as the fifth DNA base containing heritable
information. Therefore, highly sensitive, accurate, and quanti-
tative information concerning cytosine methylation in DNA
would be valuable with respect to genetic disease diagnosis.
Two major cytosine methylation assay methods have been
reported. One is hydrolysis and sequencing with a bisulfite
salt,5,6 and the other is a cleavage assay with methyl-cytosine
sensitive (or insensitive) restriction enzymes.7 A bisulfite based
determination method is very widely used to distinguish
between cytosine and methyl-cytosine. Treatment with bisulfite
converts cytosine to uracil, while methyl-cytosine remains
unaffected. Therefore, information about methyl-cytosine in
DNA can be obtained by combining bisulfite treatment and a
polymer.
This document summarizes in vitro experiments evaluating the anti-diabetic effects of alkaloidal fractions from Tinospora cordifolia and pentacyclic acid triterpenoids. Rat insulinoma cells were treated with fractions to measure insulin secretion. The fractions inhibited PTP-1B enzyme activity and glucose production in hepatocytes in a concentration-dependent manner, indicating anti-diabetic effects. Molecular docking suggested the compounds bind to a secondary site on PTP-1B, inhibiting the enzyme by a mixed inhibition mechanism. The results suggest pentacyclic triterpenoids may have potential as insulin sensitizers for treating type 2 diabetes.
This document summarizes different bioconjugation chemistries used to prepare antibody-drug conjugates (ADCs), including lysine-mediated and cysteine-mediated conjugation. For lysine-mediated conjugation of an IgG1 antibody, the average drug-to-antibody ratio was 4.3 with 2.5% unconjugated antibody and 3.9% aggregates. For cysteine-mediated conjugation using a non-cleavable linker-payload, parameters like the stoichiometry of the reducing agent TCEP and linker-payload, reaction pH, antibody concentration, and buffer concentration were evaluated. Higher TCEP amounts increased drug loading but reduced aggregates and unconjugated antibody. Optimal pH was 6
The document analyzes mutant strains of the bacterium Clostridium thermocellum to investigate how genetic alterations affected metabolism. Three mutant strains (LL1138, LL1163, LL1251) and a wild type strain (LL345) were cultured and analyzed. Isotope labeling experiments showed the mutant strains had differing levels of reversibility in glycolytic reactions compared to the wild type. LL1163 exhibited higher concentrations of GDP and GTP, suggesting altered pathways for converting phosphoenolpyruvate to pyruvate. Further experiments expressing new enzymes could engineer more efficient metabolic pathways.
This document provides information on various histological staining techniques used to highlight different tissue components. It begins by describing hematoxylin and eosin staining as the basic staining method. It then categorizes and describes special staining techniques used to identify carbohydrates, nucleic acids, lipids, amyloid, microorganisms, and connective tissues. For each category, it lists the specific stains and reagents used and the tissue components that are highlighted by each stain. The document serves as a comprehensive reference for histological staining methods.
Abstract
Aberrant mucin-type O-glycosylation by glycosyltransferases is a well-described hallmark of many cancers and is also associated with additional non-cancerous developmental and metabolic disorders. The current review focuses on N-acetylgalactosaminyltransferase genes (GALNT) and proteins (GalNAcTs) to illustrate their importance in cancer biology. Aberrant O-glycosylation by GalNAcTs activates a wide range of proteins that carry out interactions of sessile and motile cells affecting organogenesis, responses to agonists and stimulating hyperproliferation and metastatisation of neoplastic cells. As genome-wide analyses have provided abundant clues regarding under- or over-expressed genes that characterize different types of cancers, GALNTs and their transferase products have attracted attention by being unexpected actors in neoplastic contexts. We intend to review the current knowledge on GALNTs and their encoded transferases in cancer and suggest what could be the significance of such information in cancer pathogenesis and management.
Similar to ASMS Conference Poster 2015_Andrey (20)
1. Figure 3: Two hours of reaction yielded [glycopeptide+38] as main products, with
lactonization in both sialylated glycoforms, as observed before esterification of glycans 5,7,8.
As the EG2‐hFc antibody was produced in CHO cells2, 9 that do not express 2,6
sialyltransferase6, lactone formation is consistent with an 2,3 linkage.
Integration values in Table 1 clearly show that MALDI-MS of esterified species provides a
higher assessment of the sialylation content than when performed on unreacted
glycopeptides.
Esterification of non mutant and mutant Tratzumab Eg2 glycopeptides. The (*) symbol
indicates the metastable peaks in Figures 4 and 5. After ethyl esterification, neutralized
sialylated glycoforms contributed to decrease the metastable fragmentation rate. The inset
in Figure 5 (bottom) shows the difference in peak shape between adjacent regular (m/z
2997) and metastable (m/z 3049) ion peaks. The mutant samples produced more of these
metastable ions that the nonmutants, due to higher contents in sialic acid.
Upon esterification, nonsialylated glycoforms saw their masses increase by 10 (+28, ‐18).
For sialylated glycoforms, an extra addition of 28 per sialic acid residue was observed.
Figure 4 shows these results for non‐mutant TZM, and Figure 5 shows comparable results
for the mutant variant of this mAb. Table 2 highlights the relative abundance comparison of
mutant TMZ2 mAb glycopeptides before and after ethyl esterification.
Spectra of the esterified species showed two forms of detection of sialic acid. In Figures 4-5,
peaks at m/z 3326 correspond to the addition of an ethyl esterified sialic acid residue to
core-fucosylated complex type bi-antennary N-glycan (G2F), suggesting a 2,6 linkage of
sialic acid to galactose. This is expected, as these mAbs were produced in the presence of
2,6 sialyltransferase3. On the other hand, m/z 3280 peaks represent an addition of 273 to
G2F, i.e. lactonization of sialic acid, indicating a 2,3 linkage. This is also expected as the
mAbs were produced in CHO cells6.
Conclusion
Esterification of mAb glycopeptides leads to one main product per glycoform, and
enabled to differentiate 2,3 from 2,6 sialyl linkages
This fast and simple method is efficient for the analysis of sialylated glycopeptides, as it
neutralizes sialic acids and decrease the metastable fragmentation rate
MALDI-MS ion abundances give a better estimate of sialylation levels for esterified that
for unreacted glycopeptides by MALDI-TOF-MS reflectron positive mode.
Antibody esterified ion patterns are simple and predictable
References
1. J. M. Hayes et al., in M. Daeron and F. Nimmerjahn (eds.), Curr. Topics Microbiol. Immunol. 382,
2014, doi: 10.1007/978-3-319-07911-0_8, Springer Intl Publishing.
2. J. Zhang et al. Protein Expr Purif 2009, 65, 77.
3. C. Raymond, et al., MAbs 2015, 7, 571.
4. P. Lopez et al., submitted.
5. K. R. Reiding et al., Anal Chem 2014, 86, 5784.
6. M. Butler, M. Spearman, Curr Opin Biotechnol 2014, 30, 107.
7. X. Liu et al., Anal Chem 2007, 79, 3894.
8. D. J. Harvey, J Am Soc Mass Spectrom 2005, 16, 647.
9. E. Bodnar et al., Can J Chem, Epub doi: 10.1139/cjc-2015-0061.
Acknowledgements
• Members of MabNet including Dr. Mike Butler’s laboratory (Microbiology, University of
Manitoba) for providing monoclonal antibodies for analysis
• Members of the Perreault research group for their help and discussion.
Second Level Head
Body text.
2013
Overview
A fast, simple and convenient glycopeptide esterification method is demonstrated here. Monoclonal
antibody (mAb) glycopeptides isolated from tryptic digests were ethyl esterified, allowing for better
estimation of sialylation levels and for differentiation of terminal sialic acid linkages by MALDI-TOF-
MS.
Introduction
Glycosylation has become an indispensable parameter to control in the production of
biopharmaceuticals. This type of post translation modification has been demonstrated to control the
efficacy of proteins by changing solubility, folding, and receptor binding activity1. Mammalian
antibody N-glycans often exhibit terminal sialic acids that are important to cellular communication,
protein half-life1 as well as providing anti-inflammatory properties.This study presents an
ethyl‐esterification method adapted to tryptic glycopeptides isolated from mAb proteolytic digestion
mixtures. Glycopeptides were ethyl esterified using 1-ethyl-3-(3 dimethylaminopropyl) carbodiimide
(EDC) and 1-hydroxybenzotriazole (HOBt) as activators in ethanolic solution5. This method allows
quantification of neutral esterified sialylated glycoforms and direct distinction of 2,3 and 2,6 sialic
acid linkages by MALDI-TOF-MS reflectron positive mode.
Materials
Esterification reagents 1-hydroxybenzotriazole (HOBt) hydrate and 1-ethyl-3-(3
dimethylaminopropyl) carbodiimide (EDC) were purchased from Sigma. Synthetic EEQYNSTYR
was prepared by solid phase peptide synthesis. Eg2-hFc antibody2 was obtained through the
Monoclonal Antibody NSERC Strategic Network (MabNet, www.mabnet.ca). Six sialylated
monoclonal antibody (mAb) samples were prepared for analysis also through MabNet3. Their mAb
framework was TrastuzumabTM (TMZ), corresponding to that of human IgG1. EEQFNSTYR and
EAQFNSTYR were obtained from tryptic digests of porcine IgG4.
Methods
High-performance liquid chromatography reverse phase: Isolation of glycopeptides was performed
using the following gradient: 95:5 water (H2O): acetonitrile (ACN) + 0.1% Trifluoroacetic acid (TFA)
held for 5 min, then to 90:10 H2O:ACN + 0.1%TFA over 5 min, and finally to 70:30 H2O:ACN +
0.1%TFA over 10 min. Fractions were collected manually, pooled, and evaporated to dryness.
Esterification: The method was adapted from conditions reported by Reiding et al. for free glycans5.
Briefly, 10 µg samples of tryptic mAb glycopeptides were reacted with EDC and HOBt, both 0.25 M
in ethanol (10 µL). The reaction proceeded at 37oC for 1 to 3 h depending on completion.
Esterification was stopped by the addition of an equal volume of acetonitrile. Samples were then
stored at -20oC.
Cleanup: Samples were eluted on C18 cartridges with 3 x 1 mL of H20 + 0.1%TFA, then 5 x 500 µl of
50:50 H20-ACN + 0.1% TFA.
Alkylation of synthetic EEQYNSTYR: To 20 g of the peptide, 10 L of 500 mM 2-Iodoacetamide
(IA) solution were added, and the mixture was left to react in the dark at 37oC for 3-4 days. Cleanup
of the alkylated sample was achieved using solid-phase extraction on C18 medium.
Mass Spectrometric Analysis: MALDI-MS and MS/MS analyses were performed using
UltrafleXtreme (Bruker Daltonics, Germany) in both positive and negative ion modes with 2,5-
dihydroxybenzoic acid (DHB) matrix.
Systematic Study of Glycopeptide Esterification for the Determination of Sialylation Levels in Antibodies
Andrey Oliveira1, Paul Lopez1, Rini Roy1, Céline Raymond2, Edward Bodnar1, Yves Durocher2 and Hélène Perreault1
University of Manitoba, Winnipeg, Canada1; Human Health Therapeutics Portfolio, National Research Council Canada, Montréal, Canada2
Discussion
MALDI MS has been widely used to determine the chemical structure of glycans, however the lability of
sialic acid residues has resulted in inaccurate quantification of sialylation levels. Positive mode ionization
tends to underestimate the amount of sialylated glycoforms due to their inherent negative charge(s).
Metastable ions are often observed in the reflector positive mode, but these cannot be used for quantitation
due to their distorted peak shapes. Negative mode tends to overestimate the levels of sialylation.
In order to overcome this MS limitation, neutralization of sialylated N-linked glycopeptides was achieved
using ethyl-esterification5.
SYNTHETIC EEQYNSTYR: As esterification targets carboxyl groups, the peptide was expected to have an
increment mass of 84 Da (28 x 3). Instead, an increase of 38 Da was observed for the main product, as
shown in Figure 1A (bottom spectrum) by ions at m/z 1227.7. This corresponds to the esterification of two
COOH groups in the peptide, along with the loss of a water molecule. The investigation of the esterified
product's structure was pursued by MS/MS, and Figure 1B compares the results obtained for precursor ions
at m/z 1189.5 (top) and 1227.7 (bottom). A suggested structure for the synthetic peptide after ethyl-
esterification is shown in Figure 1A. Most MS/MS matched this structure, especially those at m/z 1070.
Esterification of N-glycopeptides from porcine IgG. Figure 2 shows the comparison between N-glycosylated
glycoforms prior (on top) and after (on bottom) ethyl esterification on different peptide backbones. An
increase of 38 m/z was observed for EEQYNSTYR whereas for EAQFNSTYR the increase was of 10 m/z.
In glycosylated EEQFNSTYR, MS/MS (not shown) indicates that the mass shift correspond to (+56, -18):
addition of ethyl groups on the second glutamic acid and C-terminal arginine along with a loss of H20 from
N-terminal glutamic acid due to cyclization.
In EAQFNSTYR, the increase of 10 m/z corresponds to (+28, -10): addition of ethyl group on the C-terminal
arginine, and dehydration of N-terminal glutamic acid through cyclization. Suggested structures for both
esterified peptides are shown in Figure 2.
Esterification of N-glycopeptides from Eg2-hFc. Figure 3 shows the products of ethyl esterification reaction
on Eg2 (antibody) EEQYNSTYR glycopeptides over time. For this sample, partial lactonization of both sialic
acids had occurred prior to esterification, which indicates α2,3 binding of sialic acid to galactose, as typically
encountered in CHO cells6. After 1 h of reaction time, an increase of 10 m/z was observed for nonsialylated
glycoform products. For sialylated glycoforms lactonization took place. These species were detected at
increments of 273 m/z relative to G2F (+291, ‐18). Ions at m/z 2616.2, 2778.3, 2940.3, 3213.4 and 3486.5
correspond to [glycopeptide+28] products having lost an ethanol molecule. They appear as minor products
but could also result from in‐source fragmentation.
Glycoform FG0 FG1 FG2 FG2S FG2S2
Unreacted + 58.3 100 83 11.4 4
Esterified + 55 100 83.6 20.8 9.4
Unreacted - 54.8 100 88.4 29.4 12.7
TABLE 1: Relative abundances of ions corresponding to glycoforms of EEQYNSTYR from EG2-hFc obtained
by MALDI-MS for unreacted and esterified (2 h) samples. The experimental error is within +/- 10% of the
reported values.
TABLE 2: Relative abundances of ions corresponding to glycoforms of EEQYNSTYR mutant TMZ2 sample
analyzed by MALDI-MS. The experimental error is within +/- 10% of the reported values.
Glycoform FG0 FG1 FG2 FG2S FG2S2
Unreacted + 3.6 11.7 100 67.7 12.7
Esterified + 0 16.2 100 258 112
Unreacted - 0 18.2 100 667.2 182.2
2997.348
3049.380
3067.2453010.105
3037.104
0.0
0.2
0.4
0.6
0.8
1.0
4x10
Intens.[a.u.]
2990 3000 3010 3020 3030 3040 3050 3060
m/z
A B
FIGURE 1: A) Positive, reflective mode MALDI-TOF-MS spectra of synthetic peptide EEQYNSTYR.
Top: before and bottom: after esterification. B) MALDI-TOF MS/MS spectra of [M+H]+ ions of top:
synthetic peptide EEQYNSTYR and bottom: ethyl-esterified EEQYNSTYR.
FIGURE 2: Positive, reflective mode MALDI-TOF-MS spectra of complex glycoforms of EEQFNSTYR and
EAQFNSTYR porcine IgG4. Top: unreacted glycopeptides and bottom: esterified glycopeptides.
FIGURE 3: Positive, reflective mode MALDI-TOFMS spectra of lightly sialylated glycoforms of EEQYNSTYR
from EG2-hfc obtained for top: unreacted glycopeptides and bottom 3: esterified glycopeptides.
FIGURE 4: Reflective more MALDI-TOFMS spectra of non-mutant TMZ2 mAb EEQYNSTYR glycoforms. Top:
positive mode, unreacted; middle: positive mode, esterified; bottom: negative mode, unreacted.
FIGURE 5: Reflective mode MALDI-TOFMS spectra of mutant TMZ2 mAb EEQYNSTYR glycoforms. Top:
positive mode, unreacted; middle: positive mode, esterified; bottom: negative mode, unreacted. Inset:
comparison of peak shapes for regular (left) and metastable (right) ions generated by the in-flight loss of sialic
acid.