The Experts below are selected from a list of 198 Experts worldwide ranked by ideXlab platform
Kalyan R. Anumula - One of the best experts on this subject based on the ideXlab platform.
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unique anthranilic acid chemistry facilitates profiling and characterization of ser thr linked sugar chains following Hydrazinolysis
Analytical Biochemistry, 2008Co-Authors: Kalyan R. AnumulaAbstract:A novel method for the analysis of Ser/Thr-linked sugar chains was made possible by the virtue of unique anthranilic acid (AA, 2-aminobenzoic acid [2AA]) chemistry for labeling carbohydrates in aqueous salt solutions (K. R. Anumula, Anal. Biochem. 350 (2006) 1-23). The protocol for profiling of Ser/Thr carbohydrates by Hydrazinolysis was made simple by eliminating intermediary isolation steps involved in a sample preparation such as desalting and various chromatographic purification schemes. A 6-h Hydrazinolysis was carried out at 60 degrees C for O-linked oligosaccharides and at 95 degrees C for total oligosaccharides (N-linked with some O-linked). Following evaporation of hydrazine (<10 min), the oligosaccharides were N-acetylated and derivatized with AA in the same reaction mixture containing salts. Presumably, the glycosyl-hydrazines/hydrazones present in the mixture did not interfere with AA labeling. Because AA is the most fluorescent and highly reactive tag for labeling carbohydrates, the procedures described are suitable for the analysis of a limited amount of samples ( approximately 5 microg) by the current high-resolution high-performance liquid chromatography (HPLC) methods. HPLC conditions developed for the separation of O-linked sugar chains based on size on an amide column were satisfactory for quantitative profiling and characterization. Common O-linked sugar chains found in fetuin, equine chorionic gonadotropin, and glycophorin can be analyzed in less than 50 min. In addition, these fast profiling methods were comparable to profiling by PNGase F (peptide N-glycosidase from Flavobacterium meningosepticum) digestion in terms of time, effort, and simplicity and also were highly reproducible for routine testing. The procedures for the release of sugar chains by Hydrazinolysis at the microgram level, labeling with fluorescent tag AA, and profiling by HPLC should be useful in characterization of carbohydrates found in glycoproteins.
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Unique anthranilic acid chemistry facilitates profiling and characterization of Ser/Thr-linked sugar chains following Hydrazinolysis
Analytical biochemistry, 2007Co-Authors: Kalyan R. AnumulaAbstract:A novel method for the analysis of Ser/Thr-linked sugar chains was made possible by the virtue of unique anthranilic acid (AA, 2-aminobenzoic acid [2AA]) chemistry for labeling carbohydrates in aqueous salt solutions (K. R. Anumula, Anal. Biochem. 350 (2006) 1-23). The protocol for profiling of Ser/Thr carbohydrates by Hydrazinolysis was made simple by eliminating intermediary isolation steps involved in a sample preparation such as desalting and various chromatographic purification schemes. A 6-h Hydrazinolysis was carried out at 60 degrees C for O-linked oligosaccharides and at 95 degrees C for total oligosaccharides (N-linked with some O-linked). Following evaporation of hydrazine (
Akira Otaka - One of the best experts on this subject based on the ideXlab platform.
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sequence independent traceless method for preparation of peptide protein thioesters using cpasey mediated Hydrazinolysis
Chemical & Pharmaceutical Bulletin, 2020Co-Authors: Masahiro Ueda, Chiaki Komiya, Akira Shigenaga, Sayuki Arii, Kohshi Kusumoto, Masaya Denda, Keiichiro Okuhira, Akira OtakaAbstract:Proteins incorporating artificial moieties such as fluorophores and drugs have enjoyed increasing use in chemical biology and drug development research. Preparation of such artificial protein derivatives has relied mainly on native chemical ligation in which peptide/protein thioesters chemoselectively react with N-terminal cysteine (Cys) peptides to afford protein molecules. The protein thioesters derived from expressed proteins represent thioesters that are very useful for the preparation of artificial proteins by native chemical ligation with synthetic peptides with N-terminal Cys. We recently have developed a traceless thioester-producing protocol using carboxypeptidase Y (CPaseY) which is compatible with an expressed protein. The traceless character is ensured by CPaseY-mediated Hydrazinolysis of C-terminal Xaa (X)-Cys-proline (Pro)-leucine (Leu)-OH sequence followed by an auto-processing of the Cys-Pro (CP) dipeptide unit, affording the corresponding X-thioester (X-SR). However, Hydrazinolysis of the amide bond in the prolyl leucine junction depends significantly on the nature of X. In the case of hydrophobic X residues, the Hydrazinolysis overreacts to give several hydrazides while the reaction of hydrophilic X residues proceeds slowly. In this research, we attempted to develop an X-independent CPaseY-mediated protocol and found that the incorporation of a triple CP sequence into the C-terminal end (X-(CP)3-Leu-OH) allows for efficient X-SR formation in a manner that is independent of X.
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Sequence-Independent Traceless Method for Preparation of Peptide/Protein Thioesters Using CPaseY-Mediated Hydrazinolysis.
Chemical & pharmaceutical bulletin, 2020Co-Authors: Masahiro Ueda, Chiaki Komiya, Akira Shigenaga, Sayuki Arii, Kohshi Kusumoto, Masaya Denda, Keiichiro Okuhira, Akira OtakaAbstract:Proteins incorporating artificial moieties such as fluorophores and drugs have enjoyed increasing use in chemical biology and drug development research. Preparation of such artificial protein derivatives has relied mainly on native chemical ligation in which peptide/protein thioesters chemoselectively react with N-terminal cysteine (Cys) peptides to afford protein molecules. The protein thioesters derived from expressed proteins represent thioesters that are very useful for the preparation of artificial proteins by native chemical ligation with synthetic peptides with N-terminal Cys. We recently have developed a traceless thioester-producing protocol using carboxypeptidase Y (CPaseY) which is compatible with an expressed protein. The traceless character is ensured by CPaseY-mediated Hydrazinolysis of C-terminal Xaa (X)-Cys-proline (Pro)-leucine (Leu)-OH sequence followed by an auto-processing of the Cys-Pro (CP) dipeptide unit, affording the corresponding X-thioester (X-SR). However, Hydrazinolysis of the amide bond in the prolyl leucine junction depends significantly on the nature of X. In the case of hydrophobic X residues, the Hydrazinolysis overreacts to give several hydrazides while the reaction of hydrophilic X residues proceeds slowly. In this research, we attempted to develop an X-independent CPaseY-mediated protocol and found that the incorporation of a triple CP sequence into the C-terminal end (X-(CP)3-Leu-OH) allows for efficient X-SR formation in a manner that is independent of X.
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traceless synthesis of protein thioesters using enzyme mediated Hydrazinolysis and subsequent self editing of the cysteinyl prolyl sequence
Chemical Communications, 2019Co-Authors: Chiaki Komiya, Akira Shigenaga, Jun Tsukimoto, Masahiro Ueda, Takuya Morisaki, Tsubasa Inokuma, Kohji Itoh, Akira OtakaAbstract:A traceless thioester-producing protocol featuring carboxypeptidase Y-mediated Hydrazinolysis of cysteinyl prolyl leucine-tagged peptides has been developed. The Hydrazinolysis followed by thioesterification affords cysteinyl prolyl thioesters. Self-editing of the tag and subsequent trans-thioesterification yields peptide thioesters. The developed protocol was successfully applied to the conversion of recombinant proteins to thioesters.
T Olczak - One of the best experts on this subject based on the ideXlab platform.
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Oligosaccharides released by Hydrazinolysis from Tamm–Horsfall protein of various human donors contain similar high-mannose glycans
Clinica Chimica Acta, 1999Co-Authors: M Olczak, T OlczakAbstract:As pathophysiological functions claimed for Tamm-Horsfall protein (THP) are related to its sugar moiety, we examined influence of pregnancy and various diseases on high-mannose chains. Hydrazinolysis was used to liberate oligosaccharides from THP polypeptide backbone. After HPLC separation of fluorescently labelled glycans similar profiles of neutral oligosaccharides were observed in THP of healthy subjects, pregnant women, patients with Bartter's syndrome, patients with acute lymphoblastic leukemia and a patient with carbohydrate deficient glycoprotein syndrome. THP contains Man-5, Man-6 and Man-7 glycans, with the preponderant amount of Man-6 glycan (about 7% of total THP oligosaccharides). No statistically significant differences were found in THP high-mannose glycans profiles between control subjects and pregnant women or patients. It is likely that neither pregnancy nor the pathological conditions examined affect high-mannose chains. In our opinion Hydrazinolysis as a method of oligosaccharides liberation, in contrast to enzymatic deglycosylation, is more appropriate for analysis of the sugar moiety of THP.
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Oligosaccharides released by Hydrazinolysis from Tamm-Horsfall protein of various human donors contain similar high-mannose glycans.
Clinica chimica acta; international journal of clinical chemistry, 1999Co-Authors: M Olczak, T OlczakAbstract:As pathophysiological functions claimed for Tamm-Horsfall protein (THP) are related to its sugar moiety, we examined influence of pregnancy and various diseases on high-mannose chains. Hydrazinolysis was used to liberate oligosaccharides from THP polypeptide backbone. After HPLC separation of fluorescently labelled glycans similar profiles of neutral oligosaccharides were observed in THP of healthy subjects, pregnant women, patients with Bartter's syndrome, patients with acute lymphoblastic leukemia and a patient with carbohydrate deficient glycoprotein syndrome. THP contains Man-5, Man-6 and Man-7 glycans, with the preponderant amount of Man-6 glycan (about 7% of total THP oligosaccharides). No statistically significant differences were found in THP high-mannose glycans profiles between control subjects and pregnant women or patients. It is likely that neither pregnancy nor the pathological conditions examined affect high-mannose chains. In our opinion Hydrazinolysis as a method of oligosaccharides liberation, in contrast to enzymatic deglycosylation, is more appropriate for analysis of the sugar moiety of THP.
Radoslaw P Kozak - One of the best experts on this subject based on the ideXlab platform.
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O-glycan patterns obtained from triplicate releases from saliva sample collected in the morning.
2016Co-Authors: Radoslaw P Kozak, Louise Royle, Daryl L Fernandes, Paulina A. Urbanowicz, Chamindie Punyadeera, Karli R. Reiding, Bas C. Jansen, Daniel I. Spencer, Manfred WuhrerAbstract:Samples were washed with 0.1% TFA followed by Hydrazinolysis, procainamide labelling and HILIC-UHPLC analysis with fluorescence detection. 18 O-glycan structures were selected for comparison.
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HILIC-UHPLC O-glycan profiles of saliva reference sample collected from a single individual in the morning.
2016Co-Authors: Radoslaw P Kozak, Louise Royle, Daryl L Fernandes, Paulina A. Urbanowicz, Chamindie Punyadeera, Karli R. Reiding, Bas C. Jansen, Daniel I. Spencer, Manfred WuhrerAbstract:Sample was buffer exchange into 0.1% TFA, and the O-glycans were released in triplicate by Hydrazinolysis followed by procainamide labelling and HILIC-UHPLC analysis with fluorescence detection.
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HILIC-UHPLC profile of saliva O-glycan patterns.
2016Co-Authors: Radoslaw P Kozak, Louise Royle, Daryl L Fernandes, Paulina A. Urbanowicz, Chamindie Punyadeera, Karli R. Reiding, Bas C. Jansen, Daniel I. Spencer, Manfred WuhrerAbstract:Samples were collected in days 1–5 in the morning. Samples were buffer exchanged into 0.1% TFA, and the O-glycans were released by Hydrazinolysis followed by procainamide labelling and HILIC-UHPLC analysis with fluorescence detection.
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improved nonreductive o glycan release by Hydrazinolysis with ethylenediaminetetraacetic acid addition
Analytical Biochemistry, 2014Co-Authors: Radoslaw P Kozak, Louise Royle, Richard A Gardne, Albe Ond, Daryl L Fernandes, Manfred WuhreAbstract:The study of protein O-glycosylation is receiving increasing attention in biological, medical, and biopharmaceutical research. Improved techniques are required to allow reproducible and quantitative analysis of O-glycans. An established approach for O-glycan analysis relies on their chemical release in high yield by Hydrazinolysis, followed by fluorescent labeling at the reducing terminus and high-performance liquid chromatography (HPLC) profiling. However, an unwanted degradation known as "peeling" often compromises Hydrazinolysis for O-glycan analysis. Here we addressed this problem using low-molarity solutions of ethylenediaminetetraacetic acid (EDTA) in hydrazine for O-glycan release. O-linked glycans from a range of different glycoproteins were analyzed, including bovine fetuin, bovine submaxillary gland mucin, and serum immunoglobulin A (IgA). The data for the O-glycans released by hydrazine with anhydrous EDTA or disodium salt dihydrate EDTA show high yields of the native O-glycans compared with the peeled product, resulting in a markedly increased robustness of the O-glycan profiling method. The presented method for O-glycan release demonstrates significant reduction in peeling and reduces the number of sample handling steps prior to release.
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Suppression of peeling during the release of O-glycans by Hydrazinolysis.
Analytical biochemistry, 2012Co-Authors: Radoslaw P Kozak, Louise Royle, Daryl L Fernandes, Richard A. Gardner, Manfred WuhrerAbstract:Abstract The analysis of O-glycans is essential for better understanding their functions in biological processes. Although many techniques for O-glycan release have been developed, the Hydrazinolysis release method is the best for producing O-glycans with free reducing termini in high yield. This release technique allows the glycans to be labeled with a fluorophore and analyzed by fluorescence detection. Under the Hydrazinolysis release conditions, a side reaction is observed and causes the loss of monosaccharides from the reducing terminus of the glycans (known as peeling). Using bovine fetuin (because it contains the sialylated O-glycans most commonly found on biopharmaceuticals) and bovine submaxillary gland mucin (BSM), here we demonstrate that peeling can be greatly reduced when the sample is buffer exchanged prior to Hydrazinolysis with solutions of either 0.1% trifluoroacetic acid (TFA) or low-molarity (100, 50, 20, and 5 mM) ethylenediaminetetraacetic acid (EDTA). The addition of calcium chloride to fetuin resulted in an increase in peeling, whereas subsequent washing with EDTA abolished this effect, suggesting a role of calcium and possibly other cations in causing peeling. The presented technique for sample preparation prior to Hydrazinolysis greatly reduces the level of undesirable cleavage products in O-glycan analysis and increases the robustness of the method.
Manfred Wuhrer - One of the best experts on this subject based on the ideXlab platform.
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HILIC-UHPLC O-glycan profiles of saliva reference sample collected from a single individual in the morning.
2016Co-Authors: Radoslaw P Kozak, Louise Royle, Daryl L Fernandes, Paulina A. Urbanowicz, Chamindie Punyadeera, Karli R. Reiding, Bas C. Jansen, Daniel I. Spencer, Manfred WuhrerAbstract:Sample was buffer exchange into 0.1% TFA, and the O-glycans were released in triplicate by Hydrazinolysis followed by procainamide labelling and HILIC-UHPLC analysis with fluorescence detection.
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O-glycan patterns obtained from triplicate releases from saliva sample collected in the morning.
2016Co-Authors: Radoslaw P Kozak, Louise Royle, Daryl L Fernandes, Paulina A. Urbanowicz, Chamindie Punyadeera, Karli R. Reiding, Bas C. Jansen, Daniel I. Spencer, Manfred WuhrerAbstract:Samples were washed with 0.1% TFA followed by Hydrazinolysis, procainamide labelling and HILIC-UHPLC analysis with fluorescence detection. 18 O-glycan structures were selected for comparison.
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HILIC-UHPLC profile of saliva O-glycan patterns.
2016Co-Authors: Radoslaw P Kozak, Louise Royle, Daryl L Fernandes, Paulina A. Urbanowicz, Chamindie Punyadeera, Karli R. Reiding, Bas C. Jansen, Daniel I. Spencer, Manfred WuhrerAbstract:Samples were collected in days 1–5 in the morning. Samples were buffer exchanged into 0.1% TFA, and the O-glycans were released by Hydrazinolysis followed by procainamide labelling and HILIC-UHPLC analysis with fluorescence detection.
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Suppression of peeling during the release of O-glycans by Hydrazinolysis.
Analytical biochemistry, 2012Co-Authors: Radoslaw P Kozak, Louise Royle, Daryl L Fernandes, Richard A. Gardner, Manfred WuhrerAbstract:Abstract The analysis of O-glycans is essential for better understanding their functions in biological processes. Although many techniques for O-glycan release have been developed, the Hydrazinolysis release method is the best for producing O-glycans with free reducing termini in high yield. This release technique allows the glycans to be labeled with a fluorophore and analyzed by fluorescence detection. Under the Hydrazinolysis release conditions, a side reaction is observed and causes the loss of monosaccharides from the reducing terminus of the glycans (known as peeling). Using bovine fetuin (because it contains the sialylated O-glycans most commonly found on biopharmaceuticals) and bovine submaxillary gland mucin (BSM), here we demonstrate that peeling can be greatly reduced when the sample is buffer exchanged prior to Hydrazinolysis with solutions of either 0.1% trifluoroacetic acid (TFA) or low-molarity (100, 50, 20, and 5 mM) ethylenediaminetetraacetic acid (EDTA). The addition of calcium chloride to fetuin resulted in an increase in peeling, whereas subsequent washing with EDTA abolished this effect, suggesting a role of calcium and possibly other cations in causing peeling. The presented technique for sample preparation prior to Hydrazinolysis greatly reduces the level of undesirable cleavage products in O-glycan analysis and increases the robustness of the method.
