A New Approach to Explore the Impact of Freeze-Thaw Cycling on Protein Structure: Hydrogen/Deuterium Exchange Mass Spectrometry (HX-MS)

Zhang, Aming; Wei, Qi; Singh, Satish K.; Fernandez, Erik J.

doi:10.1007/s11095-011-0383-z

Cited by 40 publications

(26 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This agreed with the high structure stability of antibody molecule against the freezing stress described above. In a previous study, we have also demonstrated that labile protein such as LDH with low structure stability to the freezing stress would accordingly produce non-native aggregates consisting of more unfolded monomers after F/T cycles (28). For antibody aggregation caused by F/T stress, similar conclusion has also been made for a different antibody in a different study (8), but the evidence was based on the analysis of global antibody structure by CD and FTIR.…”

Section: Discussionmentioning

confidence: 60%

“…The impact of freezing on Bevacizumab structure integrity was analyzed using the same HX-MS protocol in the frozen state as previously described (28). Figure 1a To further examine antibody stability against freezing stress, we increased the denaturing stress in the frozen state by adding 25 to 200 mM guanidine HCl to the initial sample solution.…”

Section: Hx Analysis Of Bevacizumab Structure In the Frozen Statementioning

confidence: 99%

See 1 more Smart Citation

Distinct Aggregation Mechanisms of Monoclonal Antibody Under Thermal and Freeze-Thaw Stresses Revealed by Hydrogen Exchange

et al. 2011

Self Cite

View full text Add to dashboard Cite

show abstract

Section: Discussionmentioning

confidence: 60%

Section: Hx Analysis Of Bevacizumab Structure In the Frozen Statementioning

confidence: 99%

Distinct Aggregation Mechanisms of Monoclonal Antibody Under Thermal and Freeze-Thaw Stresses Revealed by Hydrogen Exchange

et al. 2011

Self Cite

View full text Add to dashboard Cite

show abstract

“…15 Several groups have examined the protein-protein interfaces of irreversible antibody aggregates (i.e., dimers and oligomers) by HX-MS. For example, Zhang et al used HX-MS analysis of purified mAb aggregates to show that Fab-Fab interactions in the CDR region were generated as part of irreversible aggregate formation caused by heat exposure. 39 In addition to HX-MS, antibody aggregates have been characterized using other higher resolution techniques. For example, Deperalta et al used hydroxyl radical footprinting to map the interface region of an antibody dimer.…”

Section: Discussionmentioning

confidence: 99%

“…[34][35][36] For example, HX-MS has been recently applied to examine the effect of various solution factors and physicochemical structural changes on the local flexibility of mAbs, including salts from the Hofmeister series, 37 pharmaceutical excipients, 38 freeze-thaw cycles, 39 methionine oxidation, 40 asparagine deamidation, 41 antibody-drug conjugation, 42 deglycosylation and glycan modifications, 43 and engineered point mutations. 44 In this study, antibody clusters were characterized by a combination of DLS and chemical cross-linking experiments to determine the effect of solution conditions on the extent of RSA for an IgG1 mAb (referred to as "mAb-C") as a function of mAb-C protein concentration (1-10 mg/mL).…”

Section: Introductionmentioning

confidence: 99%

Hydrogen exchange mass spectrometry reveals protein interfaces and distant dynamic coupling effects during the reversible self-association of an IgG1 monoclonal antibody

Arora

Hickey

Majumdar

et al. 2015

mAbs

View full text Add to dashboard Cite

Volkin (2015) Hydrogen exchange mass spectrometry reveals protein interfaces and distant dynamic coupling effects during the reversible self-association of an IgG1 monoclonal antibody, mAbs, 7:3, 525-539, DOI: 10.1080DOI: 10. /19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 There is a need for new analytical approaches to better characterize the nature of the concentration-dependent, reversible self-association (RSA) of monoclonal antibodies (mAbs) directly, and with high resolution, when these proteins are formulated as highly concentrated solutions. In the work reported here, hydrogen exchange mass spectrometry (HX-MS) was used to define the concentration-dependent RSA interface, and to characterize the effects of association on the backbone dynamics of an IgG1 mAb (mAb-C). Dynamic light scattering, chemical cross-linking, and solution viscosity measurements were used to determine conditions that caused the RSA of mAb-C. A novel HX-MS experimental approach was then applied to directly monitor differences in local flexibility of mAb-C due to RSA at different protein concentrations in deuterated buffers. First, a stable formulation containing lyoprotectants that permitted freeze-drying of mAb-C at both 5 and 60 mg/mL was identified. Upon reconstitution with RSA-promoting deuterated solutions, the low vs. high protein concentration samples displayed different levels of solution viscosity (i.e., approx. 1 to 75 mPa.s). The reconstituted mAb-C samples were then analyzed by HX-MS. Two specific sequences covering complementarity-determining regions CDR2H and CDR2L (in the variable heavy and light chains, respectively) showed significant protection against deuterium uptake (i.e., decreased hydrogen exchange). These results define the major protein-protein interfaces associated with the concentration-dependent RSA of mAb-C. Surprisingly, certain peptide segments in the V H domain, the constant domain (C H 2), and the hinge region (C H 1-C H 2 interface) concomitantly showed significant increases in local flexibility at high vs. low protein concentrations. These results indicate the presence of longer-range, distant dynamic coupling effects within mAb-C occurring upon RSA.

show abstract

“…We were concerned with perturbing sample equilibrium with the nitrogen gas, as air-water interfaces can be highly denaturing [43]. Freezing can also influence protein structure [44]. Here, we used small samples in thin-walled capillaries, coupled with rapid immersion in liquid nitrogen to achieve rates of freezing that are primarily limited by the insulating properties of the silica and the Leidenfrost effect, which creates an insulating layer of nitrogen gas between the cryogenic liquid and the sample [45].…”

Section: Observations and An Improved Configurationmentioning

confidence: 99%

Amino Acid Insertion Frequencies Arising from Photoproducts Generated Using Aliphatic Diazirines

Ziemianowicz

Bomgarden

Etienne

et al. 2017

J. Am. Soc. Mass Spectrom.

View full text Add to dashboard Cite

Abstract. Mapping proteins with chemical reagents and mass spectrometry can generate a measure of accessible surface area, which in turn can be used to support the modeling and refinement of protein structures. Photolytically generated carbenes are a promising class of reagent for this purpose. Substituent effects appear to influence surface mapping properties, allowing for a useful measure of design control. However, to use carbene labeling data in a quantitative manner for modeling activities, we require a better understanding of their inherent amino acid reactivity, so that incorporation data can be normalized. The current study presents an analysis of the amino acid insertion frequency of aliphatic carbenes generated by the photolysis of three different diazirines: 3,3'-azibutyl-1-ammonium, 3,3'-azibutan-1-ol, and 4,4'-azipentan-1-oate. Leveraging an improved photolysis system for single-shot labeling of sub-microliter frozen samples, we used EThCD to localize insertion products in a large population of labeled peptides. Counting statistics were drawn from data-dependent LC-MS 2 experiments and used to estimate the frequencies of insertion as a function of amino acid. We observed labeling of all 20 amino acids over a remarkably narrow range of insertion frequencies. However, the nature of the substituent could influence relative insertion frequencies, within a general preference for larger polar amino acids. We confirm a large (6-fold) increase in labeling yield when carbenes were photogenerated in the solid phase (77 K) relative to the liquid phase (293 K), and we suggest that carbene labeling should always be conducted in the frozen state to avoid information loss in surface mapping experiments.

show abstract

A New Approach to Explore the Impact of Freeze-Thaw Cycling on Protein Structure: Hydrogen/Deuterium Exchange Mass Spectrometry (HX-MS)

Cited by 40 publications

References 45 publications

Distinct Aggregation Mechanisms of Monoclonal Antibody Under Thermal and Freeze-Thaw Stresses Revealed by Hydrogen Exchange

Distinct Aggregation Mechanisms of Monoclonal Antibody Under Thermal and Freeze-Thaw Stresses Revealed by Hydrogen Exchange

Hydrogen exchange mass spectrometry reveals protein interfaces and distant dynamic coupling effects during the reversible self-association of an IgG1 monoclonal antibody

Amino Acid Insertion Frequencies Arising from Photoproducts Generated Using Aliphatic Diazirines

Contact Info

Product

Resources

About