Characterization of Morphine–Glucose-6-phosphate Dehydrogenase Conjugates by Mass Spectrometry

Chiu, May L.; Loo, Rachel R. Ogorzalek; Loo, Joseph A.; Monbouquette, Harold G.

doi:10.1021/bc2001352

Cited by 6 publications

(6 citation statements)

References 25 publications

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“…Since the modification reaction was random and undirectional, a high conjugation degree was necessary to ensure the activity repression even though the activity of CG-G6PD conjugates might be cut down. This result agreed to the previous study [15].…”

Section: Effects Of Cg On the Residual Activity Of Cg-g6pd And The Repression Of G6pd Activity (Cg-g6pd Conjugates Were Prepared In Pbs Bsupporting

confidence: 94%

“…These results indicated that G6PD was successfully labeled with CG, and the degree of conjugation increased with CG. Recently, another work also suggested the dependence of conjugation degree on CG amount [15]. In Fig.…”

Section: Identification Of the Conjugates 311 Sodium Dodecyl Sulfate-polyacrylamide Gel Electrophoresismentioning

confidence: 90%

“…So far, EMIT has already attracted a lot of research interests in chemical analysis, especially in therapeutic drug monitoring [12] and drugs of abuse testing [14]. However, a serious decrease in the activity of reporter enzyme (up to 90%) was observed after labeling with hapten/analyte [15,16], which blocked the development of EMIT assay. Therefore, it is necessary to investigate the parameters influencing the enzyme activity.…”

Section: Fig 1 Scheme Of (A) Cg-g6pd Activity and (B) The Repression Of Cg-g6pd Activity Upon Cg Antibody Bindingmentioning

confidence: 99%

“…In EMIT assay, the reporter enzyme often loses its activity seriously after highly coupling with hapten due to the random conjugation [12,15]. Generally, the conjugation degree increases with the increase in the adding amount of hapten.…”

Section: Effects Of Cg Amountmentioning

confidence: 99%

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Fabricating cholyglycine–glucose‐6‐phosphate dehydrogenase conjugates for cholyglycine detection

Yang

Zhang

Zhi

et al. 2019

Biotech and App Biochem

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To establish cholyglycine (CG) detection via enzyme‐multiplied immunoassay technique (EMIT), glucose‐6‐phosphate dehydrogenase (G6PD) was used as a reporter enzyme to prepare hapten–enzyme conjugate. Gel electrophoresis and UV scanning demonstrated that G6PD was successfully labeled with cholyglycine, and CG‐G6PD conjugate was obtained. Furthermore, the effects of various parameters on the preparation of CG‐G6PD conjugates were investigated. Consequently, CG amount, nicotinamide adenine dinucleotide, d‐glucose‐6‐phosphate (G6P), phosphate buffer and the pH, and ionic strength of solution had important effects on the residual activity of CG‐G6PD. Moreover, CG amount, the pH, and G6P played important roles in changing CG labeling location on G6PD. Using the CG‐G6PD conjugate as test kit, the cholyglycine–EMIT calibration curve was established, which could be employed in clinical detection of cholyglycine. This study provides some valuable information for preparing hapten–G6PD conjugates.

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Section: Effects Of Cg On the Residual Activity Of Cg-g6pd And The Repression Of G6pd Activity (Cg-g6pd Conjugates Were Prepared In Pbs Bsupporting

confidence: 94%

Section: Identification Of the Conjugates 311 Sodium Dodecyl Sulfate-polyacrylamide Gel Electrophoresismentioning

confidence: 90%

Section: Fig 1 Scheme Of (A) Cg-g6pd Activity and (B) The Repression Of Cg-g6pd Activity Upon Cg Antibody Bindingmentioning

confidence: 99%

Section: Effects Of Cg Amountmentioning

confidence: 99%

See 2 more Smart Citations

Fabricating cholyglycine–glucose‐6‐phosphate dehydrogenase conjugates for cholyglycine detection

Yang

Zhang

Zhi

et al. 2019

Biotech and App Biochem

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“…A similar strategy working backward to deduce amino acid sequence containing the conjugation site has been adopted in other studies as well. 30 (3) Optionally, refine the MASCOT searching criteria to expand the variable modification of the pharmacophore-linker to additional residues identified from step 2. The final parameter setting can be generalized to perform a straightforward MASCOT search on similar conjugates.…”

Section: ■ Resultsmentioning

confidence: 99%

Development of a Potential High-Throughput Workflow to Characterize Sites of Bioconjugation by Immuno-Affinity Capture Coupled to MALDI-TOF Mass Spectrometry

et al. 2012

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The characterization of conjugation sites in bioconjugates is critical in the early discovery phase because site-specific conjugation improves in vivo stability and drug efficacy. We previously developed an engineered monoclonal antibody (mAb) scaffold which enables site-specific conjugation toward a reactive lysine (Lys) residue on each heavy chain (HC) by using an azetidinone (AZD) linker. In order to explore conjugations in other location which avoids potential interference with target binding, other chemical linkers have been studied and the investigation of N-hydroxysuccinimade (NHS) linker is reported here. The complexity of identifying the sites lies in part to the large number of Lys residues available for conjugation on the mAb scaffold. This has posed technical challenges to standard peptide mapping approaches. Therefore, an alternative strategy intended for a rapid analysis has been investigated by coupling immuno-affinity capture to matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). In this study, we have employed a novel application of two different capture formats: Surface enhanced laser dissociation/ionization (SELDI) and mass spectrometry immunoassay (MSIA) tips to reduce the analysis time. An antibody against the pharmacophore portion was immobilized to capture the conjugated peptides, and subsequently provide characterization of the conjugation sites on the scaffold. Multiple sites for the AZD and NHS linkers have been easily identified and confirmed by MS2 sequencing. Lysine99 is the predominant site for the AZD linker, and Lysine55 is the primary site for the NHS linker with Lysine193 and Tyrosine37 being minor sites as shown in the abstract figure. We have also demonstrated the use of conjugation mapping to compare the distribution pattern between the AZD and NHS linkers as well as to study the stability of conjugation sites in a rapid way.

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Withdrawal: Yang, M.‐N., Zhang, Y.‐F., Zhi, G.‐Y., Gu, X.‐F., Han, L. and Zhang, D‐H. (2020), Fabricating cholyglycine‐glucose‐6‐phosphate dehydrogenase conjugates for cholyglycine detection. Biotechnology and Applied Biochemistry. (Accepted Article): https://doi.org/10.1002/bab.1983.

Yang

Zhang

Zhi

et al. 2024

Biotech and App Biochem

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To establish cholyglycine (CG) detection via enzyme multiplied immunoassay technique (EMIT), glucose‐6‐phosphate dehydrogenase (G6PD) was used as a reporter enzyme to prepare hapten‐enzyme conjugate. Gel electrophoresis and UV scanning demonstrated that G6PD was successfully labeled with cholyglycine and CG‐G6PD conjugate was obtained. Furthermore, the effects of various parameters on the preparation of CG‐G6PD conjugates were investigated. Consequently, CG amount, NADH, D‐glucose‐6‐phosphate (G6P), phosphate buffer and the pH, and ionic strength of solution had important effects on the residual activity of CG‐G6PD. Moreover, CG amount, the pH, and G6P played important roles in changing CG labeling location on G6PD. Using the CG‐G6PD conjugate as test kit, the cholyglycine‐EMIT calibration curve was established, which could be employed in clinical detection of cholyglycine. This study provides some valuable information for preparing hapten‐G6PD conjugates.This article is protected by copyright. All rights reserved

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Characterization of Morphine–Glucose-6-phosphate Dehydrogenase Conjugates by Mass Spectrometry

Cited by 6 publications

References 25 publications

Fabricating cholyglycine–glucose‐6‐phosphate dehydrogenase conjugates for cholyglycine detection

Fabricating cholyglycine–glucose‐6‐phosphate dehydrogenase conjugates for cholyglycine detection

Development of a Potential High-Throughput Workflow to Characterize Sites of Bioconjugation by Immuno-Affinity Capture Coupled to MALDI-TOF Mass Spectrometry

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