Nonenzymatic Posttranslational Modifications and Peptide Cleavages Observed in Peptide Epimers.

Posttranslational modifications (PTMs) play vital roles in cellular homeostasis and are implicated in various pathological conditions. This work uses two ion mobility spectrometry-mass spectrometry (IMS-MS) modalities, drift-tube IMS (DT-IMS) and trapped IMS (TIMS), to characterize three important nonenzymatic PTMs that induce no mass loss: l/d isomerization, aspartate/isoaspartate isomerization, and cis/trans proline isomerization. These PTMs are assessed in a single peptide system, the recently discovered pleurin peptides, Plrn2, from Aplysia californica. We determine that the DT-IMS-MS/MS can capture and locate asparagine deamidation into aspartate and its subsequent isomerization to isoaspartate, a key biomarker for age-related diseases. Additionally, nonenzymatic peptide cleavage via in-source fragmentation is evaluated for differences in the intensities and patterns of fragment peaks between these PTMs. Peptide fragments resulting from in-source fragmentation, preceded by peptide denaturation by liquid chromatography (LC) mobile phase, exhibited cis/trans proline isomerization. Finally, the effects of differing the fragmentation voltage at the source and solution-based denaturation conditions on in-source fragmentation profiles are evaluated, confirming that LC denaturation and in-source fragmentation profoundly impact N-terminal peptide bond cleavages of Plrn2 and the structures of their fragment ions. With that, LC-IMS-MS/MS coupled with in-source fragmentation could be a robust method to identify three important posttranslational modifications: l/d isomerization, Asn-deamidation leading to Asp/IsoAsp isomerization, and cis/trans proline isomerization.

Metal migration and subunit swapping in ALS-linked SOD1: Zn2+ transfer between mutant and wild-type occurs faster than the rate of heterodimerization.

The heterodimerization of WT Cu, Zn superoxide dismutase-1 (SOD1), and mutant SOD1 might be a critical step in the pathogenesis of SOD1-linked amyotrophic lateral sclerosis (ALS). Rates and free energies of heterodimerization (ΔGHet) between WT and ALS-mutant SOD1 in mismatched metalation states-where one subunit is metalated and the other is not-have been difficult to obtain. Consequently, the hypothesis that under-metalated SOD1 might trigger misfolding of metalated SOD1 by "stealing" metal ions remains untested. This study used capillary zone electrophoresis and mass spectrometry to track heterodimerization and metal transfer between WT SOD1, ALS-variant SOD1 (E100K, E100G, D90A), and triply deamidated SOD1 (modeled with N26D/N131D/N139D substitutions). We determined that rates of subunit exchange between apo dimers and metalated dimers-expressed as time to reach 30% heterodimer-ranged from t30% = 67.75 ± 9.08 to 338.53 ± 26.95 min; free energies of heterodimerization ranged from ΔGHet = -1.21 ± 0.31 to -3.06 ± 0.12 kJ/mol. Rates and ΔGHet values of partially metalated heterodimers were more similar to those of fully metalated heterodimers than apo heterodimers, and largely independent of which subunit (mutant or WT) was metal-replete or metal-free. Mass spectrometry and capillary electrophoresis demonstrated that mutant or WT 4Zn-SOD1 could transfer up to two equivalents of Zn2+ to mutant or WT apo-SOD1 (at rates faster than the rate of heterodimerization). This result suggests that zinc-replete SOD1 can function as a chaperone to deliver Zn2+ to apo-SOD1, and that WT apo-SOD1 might increase the toxicity of mutant SOD1 by stealing its Zn2+.

Characterization of Asparagine Deamidation in Immunodominant Myelin Oligodendrocyte Glycoprotein Peptide Potential Immunotherapy for the Treatment of Multiple Sclerosis.

Mannan (polysaccharide) conjugated with a myelin oligodendrocyte glycoprotein (MOG) peptide, namely (KG)5MOG35-55, represents a potent and promising new approach for the immunotherapy of Multiple Sclerosis (MS). The MOG35-55 epitope conjugated with the oxidized form of mannan (poly-mannose) via a (KG)5 linker was found to inhibit the symptoms of MOG35-55-induced experimental autoimmune encephalomyelitis (EAE) in mice using prophylactic and therapeutic vaccinated protocols. Deamidation is a common modification in peptide and protein sequences, especially for Gln and Asn residues. In this study, the structural solution motif of deaminated peptides and their functional effects in an animal model for MS were explored. Several peptides based on the MOG35-55 epitope have been synthesized in which the Asn53 was replaced with Ala, Asp, or isoAsp. Our results demonstrate that the synthesized MOG peptides were formed to the deaminated products in basic conditions, and the Asn53 was mainly modified to Asp. Moreover, both peptides (wild type and deaminated derivative) conjugated with mannan (from Saccharomyces cerevisiae) independently inhibited the development of neurological symptoms and inflammatory demyelinating spinal cord lesions in MOG35-55-induced EAE. To conclude, mannan conjugated with a deamidated product did not affect the efficacy of the parent peptide.

Catalytic activity regulation through post-translational modification: the expanding universe of protein diversity.

Protein composition is restricted by the genetic code to a relatively small number of natural amino acids. Similarly, the known three-dimensional structures adopt a limited number of protein folds. However, proteins exert a large variety of functions and show a remarkable ability for regulation and immediate response to intracellular and extracellular stimuli. To some degree, the wide variability of protein function can be attributed to the post-translational modifications. Post-translational modifications have been observed in all kingdoms of life and give to proteins a significant degree of chemical and consequently functional and structural diversity. Their importance is partly reflected in the large number of genes dedicated to their regulation. So far, hundreds of post-translational modifications have been observed while it is believed that many more are to be discovered along with the technological advances in sequencing, proteomics, mass spectrometry and structural biology. Indeed, the number of studies which report novel post translational modifications is getting larger supporting the notion that their space is still largely unexplored. In this review we explore the impact of post-translational modifications on protein structure and function with emphasis on catalytic activity regulation. We present examples of proteins and protein families whose catalytic activity is substantially affected by the presence of post translational modifications and we describe the molecular basis which underlies the regulation of the protein function through these modifications. When available, we also summarize the current state of knowledge on the mechanisms which introduce these modifications to protein sites.

Raptor-Mediated Proteasomal Degradation of Deamidated 4E-BP2 Regulates Postnatal Neuronal Translation and NF-κB Activity.

The translation initiation repressor 4E-BP2 is deamidated in the brain on asparagines N99/N102 during early postnatal brain development. This post-translational modification enhances 4E-BP2 association with Raptor, a central component of mTORC1 and alters the kinetics of excitatory synaptic transmission. We show that 4E-BP2 deamidation is neuron specific, occurs in the human brain, and changes 4E-BP2 subcellular localization, but not its disordered structure state. We demonstrate that deamidated 4E-BP2 is ubiquitinated more and degrades faster than the unmodified protein. We find that enhanced deamidated 4E-BP2 degradation is dependent on Raptor binding, concomitant with increased association with a Raptor-CUL4B E3 ubiquitin ligase complex. Deamidated 4E-BP2 stability is promoted by inhibiting mTORC1 or glutamate receptors. We further demonstrate that deamidated 4E-BP2 regulates the translation of a distinct pool of mRNAs linked to cerebral development, mitochondria, and NF-κB activity, and thus may be crucial for postnatal brain development in neurodevelopmental disorders, such as ASD.

Deamidation and isomerization liability analysis of 131 clinical-stage antibodies.

Contemporary in vivo and in vitro discovery platform technologies greatly increase the odds of identifying high-affinity monoclonal antibodies (mAbs) towards essentially any desired biologically relevant epitope. Lagging discovery throughput is the ability to select for highly developable mAbs with drug-like properties early in the process. Upstream consideration of developability metrics should reduce the frequency of failures in later development stages. As the field moves towards incorporating biophysical screening assays in parallel to discovery processes, similar approaches should also be used to ensure robust chemical stability. Optimization of chemical stability in the early stages of discovery has the potential to reduce complications in formulation development and improve the potential for successful liquid formulations. However, at present, our knowledge of the chemical stability characteristics of clinical-stage therapeutic mAbs is fragmented and lacks comprehensive comparative assessment. To address this knowledge gap, we produced 131 mAbs with amino acid sequences corresponding to the variable regions of clinical-stage mAbs, subjected these to low and high pH stresses and identified the resulting modifications at amino acid-level resolution via tryptic peptide mapping. Among this large set of mAbs, relatively high frequencies of asparagine deamidation events were observed in CDRs H2 and L1, while CDRs H3, H2 and L1 contained relatively high frequencies of instances of aspartate isomerization.

High-throughput screening of antibody variants for chemical stability: identification of deamidation-resistant mutants.

Developability assessment of therapeutic antibody candidates assists drug discovery by enabling early identification of undesirable instabilities. Rapid chemical stability screening of antibody variants can accelerate the identification of potential solutions. We describe here the development of a high-throughput assay to characterize asparagine deamidation. We applied the assay to identify a mutation that unexpectedly stabilizes a critical asparagine. Ninety antibody variants were incubated under thermal stress in order to induce deamidation and screened for both affinity and total binding capacity. Surprisingly, a mutation five residues downstream from the unstable asparagine greatly reduced deamidation. Detailed assessment by LC-MS analysis confirmed the predicted improvement. This work describes both a high-throughput method for antibody stability screening during the early stages of antibody discovery and highlights the value of broad searches of antibody sequence space.

Structure Based Prediction of Asparagine Deamidation Propensity in Monoclonal Antibodies.

Identification of asparagine (Asn) sites that are prone to deamidation is critical for the development of therapeutic monoclonal antibodies (mAbs). Despite a common chemical degradation pathway, the rates of Asn deamidation can vary dramatically among different sites, and prediction of the sensitive deamidation sites is still challenging. In this study, characterization of Asn deamidation for five IgG1 and five IgG4 mAbs under both normal and stressed conditions revealed dramatic differences in the Asn deamidation rates. A comprehensive analysis of the deamidation sites indicated that the deamidation rate differences could be explained by differences in the local structure conformation, structure flexibility and solvent accessibility. A decision tree was developed to predict the deamidation propensity for all Asn sites in IgG mAbs based on the analysis of these three structural parameters. This decision tree will allow potential Asn deamidation hot spots to be identified early in development.

Insights into the generation of monoclonal antibody acidic charge variants during Chinese hamster ovary cell cultures.

Charge variation is one of the most important heterogeneities during monoclonal antibody (mAb) manufacturing and this study presents insights into the generation of acidic charge variants during cell culture processes. Since acidic variants generate both intracellularly and extracellularly, main charge fraction collected by weak cation exchange chromatography (WCX) was incubated in harvested cell supernatant (HCS) to simulate and investigate the extracellular process firstly. It is found that the main fraction was degraded rapidly into acidic variants rather than basic variants extracellularly, and the degradation sites were located in both Fab and Fc fragments indicated by papain digestion. Besides, certain process parameters were investigated as their potential roles in the extracellular process. As a result, media composition showed significant influence on degradation while culture time point did not, suggesting that the extracellular process was a spontaneous process without enzyme catalysis. Additionally, kinetics study reveals that the extracellular process was a pseudo first-order reaction. The E app value (21.59 kcal/mol) estimated from the Arrhenius equation suggests that the extracellular degradation might be mainly attributed to asparagine deamidation. Furthermore, we established an acidic variants generation model, indicating that the extracellular process plays a dominant role in modulating the final acidic variant level. This study provides better understanding for controlling product heterogeneity in mAb manufacturing.

Quantification of in vivo site-specific Asp isomerization and Asn deamidation of mAbs in animal serum using IP-LC-MS.

BACKGROUND: Isomerization of aspartic acid and deamidation of asparagine are two common amino acid modifications that are of particular concern if located within the complementarity-determining region of therapeutic antibodies. Questions arise as to the extent of modification occurring in circulation due to potential exposure of the therapeutic antibody to different pH regimes. RESULTS: To enable evaluation of site-specific isomerization and deamidation of human mAbs in vivo, immunoprecipitation (IP) has been combined with LC-MS providing selective enrichment, separation and detection of naive and modified forms of tryptic peptides comprising complementarity-determining region sequences. CONCLUSION: IP-LC-MS can be applied to simultaneously quantify in vivo drug concentrations and measure the extent of isomerization or deamidation in PK studies conducted during the drug discovery stage.

Independent highly sensitive characterization of asparagine deamidation and aspartic acid isomerization by sheathless CZE-ESI-MS/MS.

Amino acids residues are commonly submitted to various physicochemical modifications occurring at physiological pH and temperature. Post-translational modifications (PTMs) require comprehensive characterization because of their major influence on protein structure and involvement in numerous in vivo process or signaling. Mass spectrometry (MS) has gradually become an analytical tool of choice to characterize PTMs; however, some modifications are still challenging because of sample faint modification levels or difficulty to separate an intact peptide from modified counterparts before their transfer to the ionization source. Here, we report the implementation of capillary zone electrophoresis coupled to electrospray ionization tandem mass spectrometry (CZE-ESI-MS/MS) by the intermediate of a sheathless interfacing for independent and highly sensitive characterization of asparagine deamidation (deaN) and aspartic acid isomerization (isoD). CZE selectivity regarding deaN and isoD was studied extensively using different sets of synthetic peptides based on actual tryptic peptides. Results demonstrated CZE ability to separate the unmodified peptide from modified homologous exhibiting deaN, isoD or both independently with a resolution systematically superior to 1.29. Developed CZE-ESI-MS/MS method was applied for the characterization of monoclonal antibodies and complex protein mixture. Conserved CZE selectivity could be demonstrated even for complex samples, and foremost results obtained showed that CZE selectivity is similar regardless of the composition of the peptide. Separation of modified peptides prior to the MS analysis allowed to characterize and estimate modification levels of the sample independently for deaN and isoD even for peptides affected by both modifications and, as a consequence, enables to distinguish the formation of l-aspartic acid or d-aspartic acid generated from deaN. Separation based on peptide modification allowed, as supported by the ESI efficiency provided by CZE-ESI-MS/MS properties, and enabled to characterize and estimate studied PTMs with an unprecedented sensitivity and proved the relevance of implementing an electrophoretic driven separation for MS-based peptide analysis.

Asparagine deamidation reduces DNA-binding affinity of the Drosophila melanogaster Scr homeodomain.

Spontaneous deamidation of asparagine is a non-enzymatic post-translational modification of proteins. Residue Asn 321 is the main site of deamidation of the Drosophila melanogaster Hox transcription factor Sex Combs Reduced (Scr). Formation of iso-aspartate, the major deamidation product, is detected by HNCACB triple-resonance NMR spectroscopy. The rate of deamidation is quantified by fitting the decay of Asn NH2 side-chain signals in a time-series of (15)N-(1)H HSQC NMR spectra. The deamidated form of Scr binds to specific DNA target sequences with reduced affinity as determined by an electrophoretic mobility shift assay.