Asn Deamidation Research Articles

Phosphate-Catalyzed Succinimide Formation from an NGR-Containing Cyclic Peptide: A Novel Mechanism for Deammoniation of the Tetrahedral Intermediate.

Spontaneous deamidation in the Asn-Gly-Arg (NGR) motif that yields an isoAsp-Gly-Arg (isoDGR) sequence has recently attracted considerable attention because of the possibility of application to dual tumor targeting. It is well known that Asn deamidation reactions in peptide chains occur via the five-membered ring succinimide intermediate. Recently, we computationally showed by the B3LYP density functional theory method, that inorganic phosphate and the Arg side chain can catalyze the NGR deamidation using a cyclic peptide, c[CH2CO–NGRC]–NH2. In this previous study, the tetrahedral intermediate of the succinimide formation was assumed to be readily protonated at the nitrogen originating from the Asn side chain by the solvent water before the release of an NH3 molecule. In the present study, we found a new mechanism for the decomposition of the tetrahedral intermediate that does not require the protonation by an external proton source. The computational method is the same as in the previous study. In the new mechanism, the release of an NH3 molecule occurs after a proton exchange between the peptide and the phosphate and conformational changes. The rate-determining step of the overall reaction course is the previously reported first step, i.e., the cyclization to form the tetrahedral intermediate.

The use of in-strip digestion for fast proteomic analysis on tear fluid from dry eye patients.

Tear is an accessible fluid for exploring biomarkers of dry eye disease. This study describes a fast proteomic method by LC-Q-orbitrap-MS analysis with in-strip digestion and investigates the tear proteome of dry eye patients. Schirmer’s strips were used for collection of tear fluid from patients. These strips were cut into pieces and directly digested with trypsin before mass spectrometry analysis. The data showed that more than 50 proteins were found in tear fluid from dry eye patients. Gene Ontology (GO) annotation showed that most of proteins were transfer/carrier proteins, hydrolyses, enzyme modulators and signaling molecules. Targeted proteomics strategy revealed that 18 proteins were differentially expressed in dry eye patients. Furthermore, it was showed that the common post-translational modification in tear proteins is deamidation of Asn.

Development of novel cyclic NGR peptide-daunomycin conjugates with dual targeting property.

Cyclic NGR peptides as homing devices are good candidates for the development of drug conjugates for targeted tumor therapy. In our previous study we reported that the Dau=Aoa-GFLGK(c[KNGRE]-GG-)-NH2 conjugate has a significant antitumor activity against both CD13+ HT-1080 human fibrosarcoma and CD13− but integrin positive HT-29 human colon adenocarcinoma cells. However, it seems that the free ε-amino group of Lys in the cycle is not necessary for the biological activity. Therefore, we developed novel cyclic NGR peptide–daunomycin conjugates in which Lys was replaced by different amino acids (Ala, Leu, Nle, Pro, Ser). The exchange of the Lys residue in the cycle simplified the cyclization step and resulted in a higher yield. The new conjugates showed lower chemostability against deamidation of Asn than the control compound, thus they had lower selectivity to CD13+ cells. However, the cellular uptake and cytotoxic effect of Dau=Aoa-GFLGK(c[NleNGRE]-GG-)-NH2 was higher in comparison to the control especially on HT-29 cells. Therefore, this conjugate is more suitable for drug targeting with dual targeting property.

Structural Characterization and Physicochemical Stability Profile of a Double Mutant Heat Labile Toxin Protein Based Adjuvant

A novel protein adjuvant double-mutant Escherichia coli heat-labile toxin, LT (R192G/L211A) or dmLT, is in preclinical and early clinical development with various vaccine candidates. Structural characterization and formulation development of dmLT will play a key role in its successful process development, scale-up/transfer, and commercial manufacturing. This work describes extensive analytical characterization of structural integrity and physicochemical stability profile of dmLT from a lyophilized clinical formulation. Reconstituted dmLT contained a heterogeneous mixture of intact holotoxin (AB5, ∼75%) and free B5 subunit (∼25%) as assessed by analytical ultracentrifugation and hydrophobic interaction chromatography. Intact mass spectrometry (MS) analysis revealed presence of Lys84 glycation near the native sugar-binding site in dmLT, and forced degradation studies using liquid chromatography-MS peptide mapping demonstrated specific Asn deamidation and Met oxidation sites. Using multiple biophysical measurements, dmLT was found most stable between pH 6.5 and 7.5 and at temperatures ≤50°C. In addition, soluble aggregates and particle formation were observed upon shaking stress. By identifying the physicochemical degradation pathways of dmLT using newly developed stability-indicating analytical methods from this study, we aim at developing more stable candidate formulations of dmLT that will minimize the formation of degradants and improve storage stability, as both a frozen bulk substance and eventually as a liquid final dosage form.

Protein asparagine deamidation prediction based on structures with machine learning methods.

Chemical stability is a major concern in the development of protein therapeutics due to its impact on both efficacy and safety. Protein “hotspots” are amino acid residues that are subject to various chemical modifications, including deamidation, isomerization, glycosylation, oxidation etc. A more accurate prediction method for potential hotspot residues would allow their elimination or reduction as early as possible in the drug discovery process. In this work, we focus on prediction models for asparagine (Asn) deamidation. Sequence-based prediction method simply identifies the NG motif (amino acid asparagine followed by a glycine) to be liable to deamidation. It still dominates deamidation evaluation process in most pharmaceutical setup due to its convenience. However, the simple sequence-based method is less accurate and often causes over-engineering a protein. We introduce structure-based prediction models by mining available experimental and structural data of deamidated proteins. Our training set contains 194 Asn residues from 25 proteins that all have available high-resolution crystal structures. Experimentally measured deamidation half-life of Asn in penta-peptides as well as 3D structure-based properties, such as solvent exposure, crystallographic B-factors, local secondary structure and dihedral angles etc., were used to train prediction models with several machine learning algorithms. The prediction tools were cross-validated as well as tested with an external test data set. The random forest model had high enrichment in ranking deamidated residues higher than non-deamidated residues while effectively eliminated false positive predictions. It is possible that such quantitative protein structure–function relationship tools can also be applied to other protein hotspot predictions. In addition, we extensively discussed metrics being used to evaluate the performance of predicting unbalanced data sets such as the deamidation case.

Role of Site-Specific Asparagine Deamidation in Islet Amyloid Polypeptide Amyloidogenesis: Key Contributions of Residues 14 and 21.

Deamidation of an asparagine residue is a spontaneous non-enzymatic post-translational modification that results in the conversion of asparagine into a mixture of aspartic acid and isoaspartic acid. This chemical conversion modulates protein conformation and physicochemical properties, which could lead to protein misfolding and aggregation. In this study, we investigated the effects of site-specific Asn deamidation on the amyloidogenicity of the aggregation-prone peptide islet amyloid polypeptide (IAPP). IAPP is a 37-residue peptidic hormone whose deposition as insoluble amyloid fibrils is closely associated with type 2 diabetes. Asn residues were successively substituted with an Asp or isoAsp, and amyloid formation was evaluated by a thioflavin T fluorescence assay, circular dichroism spectroscopy, atomic force microscopy, and transmission electron microscopy. Whereas deamidation at position 21 inhibited IAPP conformational conversion and amyloid formation, the N14D mutation accelerated self-assembly and led to the formation of long and thick amyloid fibrils. In contrast, IAPP was somewhat tolerant to the successive deamidation of Asn residues 22, 31, and 35. Interestingly, a small molar ratio of IAPP deamidated at position 14 promoted the formation of nucleating species and the elongation from unmodified IAPP. Besides, using the rat pancreatic β cell line INS-1E, we observed that site-specific deamidation did not significantly alter IAPP-induced toxicity. These data indicate that Asn deamidation can modulate IAPP amyloid formation and fibril morphology and that the site of modification plays a critical role. Above all, this study reinforces the notion that IAPP amyloidogenesis is governed by precise intermolecular interactions involving specific Asn side chains.

Understanding the Diversity and Distribution of Cyclotides from Plants of Varied Genetic Origin.

Cyclotides are a large family of naturally occurring plant-derived macrocyclic cystine-knot peptides, with more than 400 having been identified in species from the Violaceae, Rubiaceae, Cucurbitaceae, Fabaceae, and Solanaceae families. Nevertheless, their specialized distribution within the plant kingdom remains poorly understood. In this study, the diversity of cyclotides was explored through the screening of 197 plants belonging to 43 different families. In total, 28 cyclotides were sequenced from 15 plant species, one of which belonged to the Rubiaceae and 14 to the Violaceae. Every Violaceae species screened contained cyclotides, but they were only sparsely represented in Rubiaceae and nonexistent in other families. The study thus supports the hypothesis that cyclotides are ubiquitous in the Violaceae, and it adds to the list of plants found to express kalata S and cycloviolacin O12. Finally, previous studies suggested the existence of cyclotide isoforms with either an Asn or an Asp at the C-terminal processing site of the cyclotide domain within the precursor proteins. Here we found that despite the discovery of a few cyclotides genuinely containing an Asp in loop 6 as evidenced by gene sequencing, deamidation of Asn during enzymatic digestion resulted in the artifactual presence of Asp isoforms. This result is consistent with studies suggesting that peptides can undergo deamidation after being subjected to external factors, including pH, temperature, and enzymatic digestion.

Succinimide Formation from an NGR-Containing Cyclic Peptide: Computational Evidence for Catalytic Roles of Phosphate Buffer and the Arginine Side Chain.

The Asn-Gly-Arg (NGR) motif and its deamidation product isoAsp-Gly-Arg (isoDGR) have recently attracted considerable attention as tumor-targeting ligands. Because an NGR-containing peptide and the corresponding isoDGR-containing peptide target different receptors, the spontaneous NGR deamidation can be used in dual targeting strategies. It is well known that the Asn deamidation proceeds via a succinimide derivative. In the present study, we computationally investigated the mechanism of succinimide formation from a cyclic peptide, c[CH2CO-NGRC]-NH2, which has recently been shown to undergo rapid deamidation in a phosphate buffer. An H2PO4− ion was explicitly included in the calculations. We employed the density functional theory using the B3LYP functional. While geometry optimizations were performed in the gas phase, hydration Gibbs energies were calculated by the SM8 (solvation model 8) continuum model. We have found a pathway leading to the five-membered ring tetrahedral intermediate in which both the H2PO4− ion and the Arg side chain act as catalyst. This intermediate, once protonated at the NH2 group on the five-membered ring, was shown to easily undergo NH3 elimination leading to the succinimide formation. This study is the first to propose a possible catalytic role for the Arg side chain in the NGR deamidation.

Preformulation Characterization, Stabilization, and Formulation Design for the Acrylodan-Labeled Glucose-Binding Protein SM4-AC

This study describes the physicochemical characterization, stabilization, and formulation design of SM4-AC, an acrylodan-labeled glucose/galactose-binding protein for use in a continuous glucose monitoring device. The physical stability profile of SM4-AC as a function of pH and temperature was monitored using a combination of biophysical techniques and the resulting physical stability profile was visualized using an empirical phase diagram. Forced degradation chemical stability studies (Asn deamidation, Met oxidation) of SM4-AC were performed using a combination of capillary isoelectric focusing, peptide mapping, and reversed-phase HPLC. Differential scanning fluorimetry was then employed to screen various pharmaceutical excipients for their ability to physically stabilize SM4-AC. An optimized formulation of 20% sucrose and 2.5 mM calcium chloride in 10 mM MES buffer, 150 mM NaCl at pH 6.0 increased the conformational stability of SM4-AC by 15°C. Accelerated and real-time stability studies were setup to compare the SM4-AC protein’s physicochemical stability and glucose-binding activity in 2 formulations for up to 12 months. SM4-AC in an optimized formulation (vs the original formulation) showed improved physical stability, and similar chemical stability and glucose binding activity profiles during storage up to 52 weeks at various temperatures.

Characterization of Glutamine Deamidation by Long-Length Electrostatic Repulsion-Hydrophilic Interaction Chromatography-Tandem Mass Spectrometry (LERLIC-MS/MS) in Shotgun Proteomics.

Deamidation of glutamine (Gln) residues is a spontaneous or enzymatic process with significant implications in aging and human pathology. Although some methods are available to identify the γ/α-glutamyl products of deamidation, none of these methods allows the characterization of this post-translational modification (PTM) from complex biological samples by shotgun proteomics. Here we present LERLIC-MS/MS, a chromatographic strategy that uses a long (50 cm) anion-exchange capillary column operating in the electrostatic repulsion-hydrophilic interaction mode (ERLIC) and coupled directly to tandem mass spectrometry (MS/MS) for proteome analysis in a single injection. Profiling of soluble extracts of brain tissues by LERLIC-MS/MS distinguished for the first time γ/α-glutamyl isomers of deamidation, encountering a 1.7 γ/α-glutamyl ratio for most Gln deamidation products. A detailed analysis of any deviation from that observed ratio allowed the identification of transglutaminase-mediated γ-glutamyl isomers as intermediate products of transamidation. Furthermore, LERLIC-MS/MS was able to simultaneously separate Gln and asparagine (Asn) deamidation products even for those peptides showing multiple deamidated proteoforms. The characterization of Asn deamidated residues by LERLIC-MS/MS also uncovered novel PIMT (protein L-isoaspartyl methyltransferase) substrate proteins in human brain tissues that deviated from the expected 3:1 isoAsp/Asp ratio. Taken together, our results demonstrate that LERLIC-MS/MS can be used to perform an in-depth study of protein deamidation on a global proteome scale. This new strategy should help to elucidate the biological implications of deamidation in aging and disease conditions.

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

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. 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. 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.

A Fluorescence-Based High-Throughput Coupled Enzymatic Assay for Quantitation of Isoaspartate in Proteins and Peptides.

Formation of isoaspartate (IsoAsp) from spontaneous asparagine (Asn) deamidation or aspartate (Asp) isomerization is one of the most common non-enzymatic pathways of chemical degradation of protein and peptide pharmaceuticals. Rapid quantitation of IsoAsp formation can enable rank-ordering of potential drug candidates, mutants, and formulations as well as support shelf life prediction and stability requirements. A coupled enzymatic fluorescence-based IsoAsp assay (CEFIA) was developed as a high-throughput method for quantitation of IsoAsp in peptides and proteins. In this note, application of this method to two therapeutic candidate proteins with distinct structural scaffolds is described. In addition, the results obtained with this method are compared to those from conventional assays.

Analysis of Monoclonal Antibody Sequence and Post-translational Modifications by Time-controlled Proteolysis and Tandem Mass Spectrometry

Methodology for sequence analysis of ∼150 kDa monoclonal antibodies (mAb), including location of post-translational modifications and disulfide bonds, is described. Limited digestion of fully denatured (reduced and alkylated) antibody was accomplished in seconds by flowing a sample in 8murea at a controlled flow rate through a micro column reactor containing immobilized aspergillopepsin I. The resulting product mixture containing 3-9 kDa peptides was then fractionated by capillary column liquid chromatography and analyzed on-line by both electron-transfer dissociation and collisionally activated dissociation mass spectrometry (MS). This approach enabled identification of peptides that cover the complete sequence of a murine mAb. With customized tandem MS and ProSightPC Biomarker search, we verified 95% amino acid residues of this mAb and identified numerous post-translational modifications (oxidized methionine, pyroglutamylation, deamidation of Asn, and several forms ofN-linked glycosylation). For disulfide bond location, native mAb is subjected to the same procedure but with longer digestion times controlled by sample flow rate through the micro column reactor. Release of disulfide containing peptides from accessible regions of the folded antibody occurs with short digestion times. Release of those in the interior of the molecule requires longer digestion times. The identity of two peptides connected by a disulfide bond is determined using a combination of electron-transfer dissociation and ion-ion proton transfer chemistry to read the two N-terminal and two C-terminal sequences of the connected peptides.

A conventional procedure to reduce Asn deamidation artifacts during trypsin peptide mapping.

Asn deamidation is a common post-translational modification of proteins with significant biological consequences. Asn deamidation can cause changes in structure, stability and function of proteins. LC-MS peptide mapping is the most widely used method to detect and quantify Asn deamidation. However, a significant amount of deamidation can occur during sample preparation for peptide mapping, making it challenging to accurately determine the original level of deamidation. Although several protocols to reduce procedure-induced deamidation have been reported, they either require special procedural steps or are not optimal for maintaining trypsin activity. In the current study, several commonly used buffers that are optimal for trypsin activity were evaluated. The results demonstrated that much lower levels of Asn deamidation artifacts were observed when Tris buffer was used, especially at lower concentrations. The addition of 10% acetonitrile further reduced the levels of Asn deamidation artifacts. The utility of the optimized procedure was demonstrated by the digestion of a recombinant monoclonal antibody. The proposed procedure can be readily applied to any laboratory settings as it does not require any special reagents or procedures.

Abstract 5434: A high through-put bioluminescent assay to monitor the deamidation of asparagine and isomerization of aspartate residues in therapeutic proteins and antibodies

Abstract Spontaneous degradation reactions such as oxidation, glycation, deamidation, Isomerization, and racemization are non-enzymatic modifications and produce functionally damaged species reflecting aging effect at the molecular level. L-asparagine (Asn) and L-aspartate (Asp) are among the most unstable residues in proteins- linked to deamidation, isomerization and racemization Rx. Under physiological conditions, succinimide-linked deamidation of Asn occurs with t1/2as short as 6 hrs while that for isoaspartate are generally 10-fold longer. A number of biological peptides and proteins possess labile Asn and Asp residues and the formation of IsoAsp at these sites adversely affect their function. The degradation of Asn and Asp sites via succinimide pathway has significant effect on protein function. Loss of function associated with isoAsp formation are found in many biologically critical proteins and is a major source of antibody instability and micro heterogeneity, Thus it is critical to accurately determine both the levels and the site of Asn deamidation in therapeutic antibodies and proteins. Although mass Spectroscopic platforms have been at the forefront of technologies to quantitate and identify sites of isoAsp formation, it fails in accurately determining Asn level and has problems with Asp isomerization due to the various steps required for sample preparation such as denaturation, reduction, alkylation, and enzyme digestion of the Ab before analysis resulting in problems with Asp isomerization and Asn determination artifacts. Although peptide mapping is the most powerful tool in isoAsp analyses, it is very time consuming and may itself actually cause sample degradation during preparation and analysis and is not always practical when large number of samples are needed for analysis. To improve on and complement existing technologies, we have developed a bioluminescent, homogenous, robust, sensitive and easy to use assay to accurately monitor the deamidation of Asn and isomerization of aspartate. The assay is based on the use of protein isoaspartate methyltransferase to catalyze the methylation of isoaspartate using S-adenosylmethionine (SAM) and resulting in the formation of S-adenosylhomocysteine and methylated isoaspartate. The assay is very sensitive, it can detect as little as 100 fmol of IsoAsp in as little as 5-10 pmol of proteins. Citation Format: Said A. Goueli, Kevin Hsiao, Rushikesh Patel, Vishal Nashine. A high through-put bioluminescent assay to monitor the deamidation of asparagine and isomerization of aspartate residues in therapeutic proteins and antibodies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5434. doi:10.1158/1538-7445.AM2015-5434

Two Decades of Publishing Excellence in Pharmaceutical Biotechnology

Recombinant biological products have revolutionized modern medicine by providing both remarkably effective vaccines to prevent disease and therapeutic drugs to treat a wide variety of unmet medical needs. Since the early 1980s, dozens of new therapeutic protein drugs and macromolecular vaccines have been commercialized, which have benefitted millions of patients worldwide. The pharmaceutical development of these biological products presented many scientific and technical challenges, some of which continue today with newer candidates including recombinant protein-based vaccines with novel adjuvants, peptide and RNA-based drugs, and stem cellular therapies. Compared with small molecule drugs, the characterization, stabilization, formulation, and delivery of biomolecules share common hurdles as well as unique challenges. This area of drug development research has been referred to as “pharmaceutical biotechnology”, in recognition of the critical role that recombinant DNA technology plays in the design and production of most of these biological products. Current research focus areas in this field include (i) determination of structural integrity of the primary sequence, post-translational modifications, and higher-order three dimensional shapes, (ii) assessment of physicochemical degradation pathways and their effects on biological activity and potency, (iii) formulation design and development to optimize stability and delivery, (iv) evaluating and optimizing process development steps including lyophilization and fill-finish, (v) analytical method development and applications of new instruments and data visualization tools, (vi) design and development of drug delivery approaches, and (vii) studies of biological effects including pharmacokinetics, pharmacodynamics, and adverse immunogenicity. During the early days of pharmaceutical biotechnology research, there were numerous scientific challenges because the analytical characterization approaches needed for development of recombinant biological molecules in “real world” pharmaceutical dosage forms were essentially unknown. Furthermore, understanding critical drug product manufacturing issues (e.g., stability of biological compounds during processing, storage, and shipping as well as reproducibility of fill-finish production technologies) and behavior during and after patient administration was often achieved by “on-the-job” training. Fortunately, the pioneers in the field regularly presented research at key conferences and started publishing early in pharmaceutical sciences journals such as Journal of Pharmaceutical Sciences. Recognizing this critically important new field, the then Editor of the journal, Professor Bill Higuchi, instituted a new “pharmaceutical biotechnology” category for research papers. This insightful move was coupled with an equally wise decision to recruit Dr. C. Russell Middaugh as the new Associate Editor for the new research category. As will be detailed below, under Dr. Middaugh’s diligent and expert guidance, pharmaceutical biotechnology papers have grown in number, scope, and impact over the past 20 years, and these days, the Journal of Pharmaceutical Sciences is viewed by scientific leaders in the field as the “go to” place for publication of the most important results and descriptions of innovations in pharmaceutical biotechnology.

Structural Characterization of Modification on the Interface between a Ligand and its Receptor for Biopharmaceuticals

The binding of a protein to its target is a major mode of action for most biopharmaceutical therapies with over 70% of biopharmaceuticals involved in binding between the protein and its target. The interfaces between a biopharmaceutical and its target are key regions for its efficacy. Any modifications to the amino acids at the interfaces invariably affect interactions between the biopharmaceutical and its receptor and may result in lowering therapeutic efficacy. Degradations of biopharmaceuticals by asparagine (Asn) deamidation and/or aspartate (Asp) isomerization have been well characterized and those modifications at the interfaces have resulted in a loss of activity. To characterize modification hot-spots on the interfaces, it is necessary to identify the amino acid residues on the interfaces. We recently addressed a visualization tool for amino acids on the interfaces between a protein ligand and its receptor. This tool was applied to visualize ligand protein-receptor interaction and antigen-antibody interaction. As a model system for ligand protein-receptor interaction, erythropoietin (EPO) and its receptor were selected and amino acids on the interfaces were identified. Modifications on the interfaces were then investigated. Deamidation of Asn was identified at two amino acid residues, Asn47 and Asp147, on Interface 1 of EPO. The relative contents of deamidated residues on the interface of EPO were in the range of 3-5% of the total. As a model system for antigen-antibody interaction, Herceptin and its receptor, HER2, were chosen and amino acids on the interfaces were identified. Then modifications on the interfaces were assessed. Deamidation of the light chain Asn30 and heavy chain Asn55 were identified. The relative contents of the deamidated residues on the interfaces were in the range of 8-9% of the total. Along with deamidation, another modification, isomerization, was identified at the amino acid residue Asp102 of the heavy chain, and the level of oxidation was 13.5% of the total. Our studies provide a targeted method focusing on the interface between a protein and its target that can be coupled with other applications, for example, identification of modified amino acids on the interfaces.

Evaluation of the effect of trypsin digestion buffers on artificial deamidation.

Nonenzymatic deamidation occurs readily under the condition of trypsin digestion, resulting in the identification of many artificial deamidation sites. To evaluate the effect of trypsin digestion buffers on artificial deamidation, we compared the three commonly used buffers Tris-HCl (pH 8), ammonium bicarbonate (ABC), and triethylammonium bicarbonate (TEAB), and ammonium acetate (pH 6), which was reported to reduce Asn deamidation. iTRAQ quantification on rat kidney tissue digested in these four buffers indicates that artificial Asn deamidation is produced in the order of ammonium acetate < Tris-HCl < ABC < TEAB, and Gln deamidation has no significant differences in all tested buffers. Label-free experiments show the same trend, while protein and unique peptide identification are comparable using these four buffers. To explain the differences of these four buffers in producing artificial Asn deamidation, we determined the half-life of Asn deamidation in these buffers using synthetic peptides containing -Asn-Gly- sequences. It is 51.4 ± 6.0 days in 50 mM of ammonium acetate (pH 6) at 37 °C, which is about 23, 104, and 137 times that in Tris-HCl, ABC, and TEAB buffers, respectively. In conclusion, ammonium acetate (pH 6) is more suitable than other tested buffers for characterizing endogenous deamidation and N-glycosylation.

Structure-Based Prediction of Asparagine and Aspartate Degradation Sites in Antibody Variable Regions

Monoclonal antibodies (mAbs) and proteins containing antibody domains are the most prevalent class of biotherapeutics in diverse indication areas. Today, established techniques such as immunization or phage display allow for an efficient generation of new mAbs. Besides functional properties, the stability of future therapeutic mAbs is a key selection criterion which is essential for the development of a drug candidate into a marketed product. Therapeutic proteins may degrade via asparagine (Asn) deamidation and aspartate (Asp) isomerization, but the factors responsible for such degradation remain poorly understood. We studied the structural properties of a large, uniform dataset of Asn and Asp residues in the variable domains of antibodies. Their structural parameters were correlated with the degradation propensities measured by mass spectrometry. We show that degradation hotspots can be characterized by their conformational flexibility, the size of the C-terminally flanking amino acid residue, and secondary structural parameters. From these results we derive an accurate in silico prediction method for the degradation propensity of both Asn and Asp residues in the complementarity-determining regions (CDRs) of mAbs.

Specific Catalysis of Asparaginyl Deamidation by Carboxylic Acids: Kinetic, Thermodynamic, and Quantitative Structure–Property Relationship Analyses

Asparaginyl (Asn) deamidation could lead to altered potency, safety, and/or pharmacokinetics of therapeutic protein drugs. In this study, we investigated the effects of several different carboxylic acids on Asn deamidation rates using an IgG1 monoclonal antibody (mAb1*) and a model hexapeptide (peptide1) with the sequence YGKNGG. Thermodynamic analyses of the kinetics data revealed that higher deamidation rates are associated with predominantly more negative ΔS and, to a lesser extent, more positive ΔH. The observed differences in deamidation rates were attributed to the unique ability of each type of carboxylic acid to stabilize the energetically unfavorable transition-state conformations required for imide formation. Quantitative structure property relationship (QSPR) analysis using kinetic data demonstrated that molecular descriptors encoding for the geometric spatial distribution of atomic properties on various carboxylic acids are effective determinants for the deamidation reaction. Specifically, the number of O-O and O-H atom pairs on carboxyl and hydroxyl groups with interatomic distances of 4-5 Å on a carboxylic acid buffer appears to determine the rate of deamidation. Collectively, the results from structural and thermodynamic analyses indicate that carboxylic acids presumably form multiple hydrogen bonds and charge-charge interactions with the relevant deamidation site and provide alignment between the reactive atoms on the side chain and backbone. We propose that carboxylic acids catalyze deamidation by stabilizing a specific, energetically unfavorable transition-state conformation of l-asparaginyl intermediate II that readily facilitates bond formation between the γ-carbonyl carbon and the deprotonated backbone nitrogen for cyclic imide formation.