Chemical crosslinking and mass spectrometric identification of interaction sites within soluble aggregate of protein therapeutics

Zhao, Alan; Hao, Gang; Gu, Jane

doi:10.1016/j.jpba.2012.05.006

Cited by 7 publications

(6 citation statements)

References 20 publications

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“…211−214 The next step in protein aggregate characterization will consist in improving the current understanding of this important biophysical process by determining interaction regions responsible for oligomerization. Zhao et al described the combination of chemical cross-linking and MS to probe the interaction among soluble oligomer formed by the Fc moeties of mAbs aggregates induced by thermal stress, 215 revealing site-specific information regarding the binding interface. Zhang et al 216 used HDX-MS, another method to gain high-resolution structural information about conformational changes and intermolecular contacts during antibody aggregation.…”

Section: ■ Dimers and Aggregatesmentioning

confidence: 99%

Characterization of Therapeutic Antibodies and Related Products

et al. 2012

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Section: ■ Dimers and Aggregatesmentioning

confidence: 99%

Characterization of Therapeutic Antibodies and Related Products

et al. 2012

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“…Gu and coworkers [114] used cross‐linking to probe the interaction of the Fc moiety of a mAb expressed in Chinese hamster Ovary cells. They formed aggregates by incubation at 40 °C for 6 h, followed by reaction with the bifunctional crosslinker BS3.…”

Section: Ms‐based Characterization Of Antibodiesmentioning

confidence: 99%

Mass spectrometry for the biophysical characterization of therapeutic monoclonal antibodies

2013

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Monoclonal antibodies (mAbs) are powerful therapeutics, and their characterization has drawn considerable attention and urgency. Unlike small-molecular drugs (150-600 Da) that have rigid structures, mAbs (~150 kDa) are engineered proteins that undergo complicated folding and can exist in a number of low-energy structures, posing a challenge for traditional methods in structural biology. Mass spectrometry (MS)-based biophysical characterization approaches can provide structural information, bringing high sensitivity, fast turnaround, and small sample consumption. This review outlines various MS-based strategies for protein biophysical characterization and then reviews how these strategies provide structural information of mAbs at the protein level (intact or top-down approaches), peptide, and residue level (bottom-up approaches), affording information on higher order structure, aggregation, and the nature of antibody complexes.

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“…We used chemical cross linking combined with mass spectrometry to identify the subunit interaction regions in αA-WT and αA-G98R crystallins. Such an approach has been successfully used to identify subunit-subunit interactions in native α-crystallin [32], [33], phage proteins [34], quaternary domain interactions in Hsp90 chaperones [35], interaction sites in soluble aggregates of monoclonal antibody [36] and sHSP21 and substrate interactions [37], etc.…”

Section: Discussionmentioning

confidence: 99%

“…The β-sandwich assembly comprising β2–β9 strands in the α-crystallin domain [38] forms an anti-parallel (AP) interface comprising the dimer, which forms the basic assembly unit for the higher order oligomerization of the wild-type protein [39]–[41]. The oligomerization involves the formation of various inter-chain interactions involving ion pairs between topologically equivalent residues [8], [36]. G98R mutation in αA-crystallin introduces a charged amino acid, which could have resulted in the gain of ion pairs in the interface not seen in WT proteins.…”

Section: Discussionmentioning

confidence: 99%

Identification of Subunit-Subunit Interaction Sites in αA-WT Crystallin and Mutant αA-G98R Crystallin Using Isotope-Labeled Cross-Linker and Mass Spectrometry

et al. 2013

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Cataract is characterized by progressive protein aggregation and loss of vision. α-Crystallins are the major proteins in the lens responsible for maintaining transparency. They exist in the lens as highly polydisperse oligomers with variable numbers of subunits, and mutations in α-crystallin are associated with some forms of cataract in humans. Because the stability of proteins is dependent on optimal subunit interactions, the structural transformations and aggregation of mutant proteins that underlie cataract formation can be understood best by identifying the residue-specific inter- and intra-subunit interactions. Chemical crosslinking combined with mass spectrometry is increasingly used to provide structural insights into intra- and inter-protein interactions. We used isotope-labeled cross-linker in combination with LC-MS/MS to determine the subunit–subunit interaction sites in cataract-causing mutant αA-G98R crystallin. Peptides cross-linked by isotope-labeled (heavy and light forms) cross-linkers appear as doublets in mass spectra, thus facilitating the identification of cross-linker–containing peptides. In this study, we cross-linked wild-type (αA-WT) and mutant (αA-G98R) crystallins using the homobifunctional amine-reactive, isotope-labeled (d0 and d4) cross-linker–BS2G (bis[sulfosuccinimidyl]glutarate). Tryptic in-solution digest of cross-linked complexes generates a wide array of peptide mixtures. Cross-linked peptides were enriched using strong cation exchange (SCX) chromatography followed by both MS and MS/MS to identify the cross-linked sites. We identified a distinct intermolecular interaction site between K88 — K99 in the β5 strand of the mutant αA-G98R crystallin that is not found in wild-type αA-crystallin. This interaction could explain the conformational instability and aggregation nature of the mutant protein that results from incorrect folding and assembly.

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Chemical crosslinking and mass spectrometric identification of interaction sites within soluble aggregate of protein therapeutics

Cited by 7 publications

References 20 publications

Characterization of Therapeutic Antibodies and Related Products

Characterization of Therapeutic Antibodies and Related Products

Mass spectrometry for the biophysical characterization of therapeutic monoclonal antibodies

Identification of Subunit-Subunit Interaction Sites in αA-WT Crystallin and Mutant αA-G98R Crystallin Using Isotope-Labeled Cross-Linker and Mass Spectrometry

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