Asparagine Deamidation Research Articles

PH/Acetonitrile-Gradient Reversed-Phase Fractionation of Enriched Hyper-Citrullinated Library in Combination with LC-MS/MS Analysis for Confident Identification of Citrullinated Peptides.

Citrullination, the Ca2+-driven enzymatic conversion of arginine residues to citrulline, is a posttranslational modification, implicated in several physiological and pathological processes. Several methods to detect citrullinated proteins have been developed, including color development reagent, fluorescence, phenylglyoxal, and antibody-based methods. These methods yet suffer from limitations in sensitivity, specificity, or citrullinated site determination. Mass spectrometry (MS)-based proteomic analysis has emerged as a promising method to resolve these problems. However, due to low abundance of citrullinated proteins and similar MS features to deamidation of asparagine and glutamine, confident identification of citrullinated proteome is challenging. Here, we present a systematic approach to identify a compendium of steps to enhance the number of detected citrullinated residue and implement diagnostic MS feature that allow the confidence of MS-based identifications. Our method is based on the concept of generation of hyper-citrullinated library with high-pH reversed-phase peptide fractionation that allows to enrich in low abundance citrullinated peptides and amplify the effect of charge loss upon citrullination. Application of our approach to complex global citrullino-proteome datasets demonstrates the confident assessment of citrullinated peptides, thereby enhancing the size and functional interpretation of citrullinated proteomes.

Deamidation drives molecular aging of the SARS-CoV-2 spike protein receptor-binding motif

The spike protein is the main protein component of the SARS-CoV-2 virion surface. The spike receptor-binding motif mediates recognition of the human angiotensin-converting enzyme 2 receptor, a critical step in infection, and is the preferential target for spike-neutralizing antibodies. Posttranslational modifications of the spike receptor-binding motif have been shown to modulate viral infectivity and host immune response, but these modifications are still being explored. Here we studied asparagine deamidation of the spike protein, a spontaneous event that leads to the appearance of aspartic and isoaspartic residues, which affect both the protein backbone and its charge. We used computational prediction and biochemical experiments to identify five deamidation hotspots in the SARS-CoV-2 spike protein. Asparagine residues 481 and 501 in the receptor-binding motif deamidate with a half-life of 16.5 and 123 days at 37 °C, respectively. Deamidation is significantly slowed at 4 °C, indicating a strong dependence of spike protein molecular aging on environmental conditions. Deamidation of the spike receptor-binding motif decreases the equilibrium constant for binding to the human angiotensin-converting enzyme 2 receptor more than 3.5-fold, yet its high conservation pattern suggests some positive effect on viral fitness. We propose a model for deamidation of the full SARS-CoV-2 virion illustrating how deamidation of the spike receptor-binding motif could lead to the accumulation on the virion surface of a nonnegligible chemically diverse spike population in a timescale of days. Our findings provide a potential mechanism for molecular aging of the spike protein with significant consequences for understanding virus infectivity and vaccine development.

Fast analysis of antibody-derived therapeutics by automated multidimensional liquid chromatography – Mass spectrometry

Characterization of post-translational modifications (PTMs) of therapeutic antibodies is commonly performed by bottom-up approaches, involving sample preparation and peptide analysis by liquid chromatography-mass spectrometry (LC-MS). Conventional sample preparation requires extensive hands-on time and can increase the risk of inducing artificial modifications as many off-line steps - denaturation, disulfide-reduction, alkylation and tryptic digestion - are performed. In this study, we developed an on-line multidimensional (mD)-LC-MS bottom-up approach for fast sample preparation and analysis of (formulated) monoclonal antibodies and antibody-derived therapeutics. This approach allows on-column reduction, tryptic digestion and subsequent peptide analysis by RP-MS. Optimization of the 1D -and 2D flow and temperature improved the trapping of small polar peptides during on-line peptide mapping analysis. These adaptations increased the sequence coverage (95–98% versus 86–94% for off-line approaches) and allowed identification of various PTMs (i.e. deamidation of asparagine, methionine oxidation and lysine glycation) within a single analysis. This workflow enables a fast (<2 h) characterization of antibody heterogeneities within a single run and a low amount of protein (10 μg). Importantly, the new mD-LC-MS bottom-up method was able to detect the polar, fast-eluting peptides: Fc oxidation at Hc-Met-252 and the Fc N-glycosylation at Hc-Asn-297, which can be challenging using mD-LC-MS. Moreover, the method showed good comparability across the different measurements (RSD of retention time in the range of 0.2–1.8% for polar peptides). The LC system was controlled by only a standard commercial software package which makes implementation for fast characterization of quality attributes relatively easy.

Energetics and mechanisms for decomposition of cationized amino acids and peptides explored using guided ion beam tandem mass spectrometry.

Fragmentation studies of cationized amino acids and small peptides as studied using guided ion beam tandem mass spectrometry (GIBMS) are reviewed. After a brief examination of the key attributes of the GIBMS approach, results for a variety of systems are examined, compared, and contrasted. Cationization of amino acids, diglycine, and triglycine with alkali cations generally leads to dissociations in which the intact biomolecule is lost. Exceptions include most lithiated species as well as a few examples for sodiated and one example for potassiated species. Like the lithiated species, cationization by protons leads to numerous dissociation channels. Results for protonated glycine, cysteine, asparagine, diglycine, and a series of tripeptides are reviewed, along with the thermodynamic consequences that can be gleaned. Finally, the important physiological process of the deamidation of asparagine (Asn) residues is explored by the comparison of five dipeptides in which the C-terminal partner (AsnXxx) is altered. The GIBMS thermochemistry is shown to correlate well with kinetic results from solution phase studies.

Cellulonodin-2 and Lihuanodin: Lasso Peptides with an Aspartimide Post-Translational Modification.

Lasso peptides are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) defined by their threaded structure. Besides the class-defining isopeptide bond, other post-translational modifications (PTMs) that further tailor lasso peptides have been previously reported. Using genome mining tools, we identified a subset of lasso peptide biosynthetic gene clusters (BGCs) that are colocalized with genes encoding protein l-isoaspartyl methyltransferase (PIMT) homologues. PIMTs have an important role in protein repair, restoring isoaspartate residues formed from asparagine deamidation to aspartate. Here we report a new function for PIMT enzymes in the post-translational modification of lasso peptides. The PIMTs associated with lasso peptide BGCs first methylate an l-aspartate side chain found within the ring of the lasso peptide. The methyl ester is then converted into a stable aspartimide moiety, endowing the lasso peptide ring with rigidity relative to its unmodified counterpart. We describe the heterologous expression and structural characterization of two examples of aspartimide-modified lasso peptides from thermophilic Gram-positive bacteria. The lasso peptide cellulonodin-2 is encoded in the genome of actinobacterium Thermobifida cellulosilytica, while lihuanodin is encoded in the genome of firmicute Lihuaxuella thermophila. Additional genome mining revealed PIMT-containing lasso peptide BGCs in 48 organisms. In addition to heterologous expression, we have reconstituted PIMT-mediated aspartimide formation in vitro, showing that lasso peptide-associated PIMTs transfer methyl groups very rapidly as compared to canonical PIMTs. Furthermore, in stark contrast to other characterized lasso peptide PTMs, the methyltransferase functions only on lassoed substrates.

Rational Development of Stable PYY3-36 Peptide Y2 Receptor Agonists.

The anorectic effect of PYY3-36 makes it a potential pharmacological weight loss treatment. Modifications of the endogenous peptide to obtain commercially attractive pharmacological and biophysical stability properties are examined. Half-life extended PYY3-36 analogues were prepared and examined regarding Y2-receptor potency as well as biophysical and stability properties. Deamidation of asparagine in position 18 and 29 was observed upon incubation at 37°C. Asparagine in position 18 - but not position 29 - could be substituted to glutamine without detrimental effects on Y2-receptor potency. Covalent dimers were formed via the phenol impurity benzoquinone reacting with two N-terminal residues (Isoleucine-Lysine). Both residues had to be modified to suppress dimerization, which could be done without negatively affecting Y2-receptor potency or other stability/biophysical properties. Introduction of half-life extending modifications in position 30 and 35 eliminated aggregation at 37°C without negatively affecting other stability properties. Placement of a protracting moiety (fatty acid) in the receptor-binding C-terminal region reduced Y2-receptor potency substantially, whereas only minor effects of protractor position were observed on structural, biophysical or stability properties. Lipidated PYY3-36 analogues formed oligomers of various sizes depending on primary structure and solution conditions. By rational design, a chemically and physically stable Y2-receptor selective, half-life extended PYY3-36 peptide has been developed.

Effect of pH and heat treatment on physicochemical and functional properties of spray-dried whey protein concentrate powder

Effects of pH and heating on deamidation of whey protein concentrate (WPC) solution and functional properties of resultant spray-dried WPC powder were investigated. Temperature and heating time affected deamidation rates with the highest reactivities for WPC solutions heated at 120 °C for 15 min and 145 °C for 120 s. Deamidation sites were pH dependent: pH 3 induced more glutamine deamidation; pH 10 induced more asparagine deamidation. The functional properties of spray-dried WPC powders were also pH dependent. WPC solution adjusted to pH 3 and heated at 145 °C for 120 s (prior to spray drying) exhibited a reduction in solubility and foamability, but markedly improved foam stability of the resultant powders; meanwhile, the properties of powders were not significantly impacted by pH adjustment to 10.0 and heating at 145 °C for 120 s. However, pH 3 and 10 with and without heating significantly improved emulsifying properties of spray-dried WPC.

Chip-Based Capillary Zone Electrophoresis Mass Spectrometry for Rapid Resolution and Quantitation of Critical Quality Attributes in Protein Biotherapeutics.

The aspiration of the multi-attribute method (MAM) is to utilize a single mass spectrometry-based method that can measure multiple attributes simultaneously, thus enabling data-driven decisions more quickly and efficiently. However, challenges associated with identifying and quantitating critical quality attributes such as asparagine deamidation and isoaspartic acid using conventional ultrahigh-pressure liquid chromatography (UHPLC) coupled to mass spectrometry have necessitated long gradients to ensure sufficient separation for quantitation. Microfluidic chip-based capillary zone electrophoresis mass spectrometry (CZE-MS) shows potential to enable rapid charge-based separation of peptide mixtures, and this approach was evaluated using multipeptide mixtures of synthetic peptides as well as digested protein therapeutics. In these experiments, repeatability, linearity, and peak-to-peak resolution of several peptide families containing asparagine deamidation and/or isoaspartic acid were demonstrated. In addition, a comparison of peptide map results acquired with both UHPLC-MS and CZE-MS for two enzymatically digested biological therapeutics showed comparable sequence coverage and quantitation results between the two approaches. As MAM becomes increasingly utilized for analysis of biological therapeutics, MS instrument demand will rapidly increase, resulting in a bottleneck. A CZE-based separation shows potential to alleviate this bottleneck by drastically increasing MAM throughput while providing results comparable to those acquired using conventional UHPLC separations.

Multiattribute Monitoring of Antibody Charge Variants by Cation-Exchange Chromatography Coupled to Native Mass Spectrometry.

The aim of this study was to characterize the product variants of a therapeutic T-cell bispecific humanized monoclonal antibody (TCB Mab, ∼200 kDa, asymmetric) and to develop an online cation-exchange chromatography native electrospray mass spectrometry method (CEC-UV-MS) for direct TCB Mab charge variant monitoring during bioprocess and formulation development. For the identification and functional evaluation of the diverse and complex TCB Mab charge variants, offline fractionation combined with comprehensive analytical testing was applied. The offline fractionation of abundant product variant peaks enabled identification of coeluting acid charge variants such as asparagine deamidation, primary and secondary Fab glycosylation (with and without sialic acid), and the presence of O-glycosylation in the G4S-linker region. Consequently, a new nonconsensus N-glycosylation motif (N-338-FG) in the heavy chain CDR region was discovered. Functional evaluation by cell-based potency testing demonstrated a clear and negative impact of both asparagine deamidations, whereas the O-glycosylation did not affect the TCB Mab biological activity. We established an online native CEC-UV-MS method, with an ammonium acetate buffer and pH gradient, to directly monitor the TCB Mab charge variants. All abundant chemical degradations and post-translational amino acid modifications already identified by offline fraction experiments and liquid chromatography mass spectrometry peptide mapping could also be monitored by the online CEC-UV-MS method. The herein reported online native CEC-UV-MS methodology represents a complementary or even alternative approach for multiattribute monitoring of biologics, offering multiple benefits, including increased throughput and reduced sample handling and intact protein information in the near-native state.

Confident Identification of Citrullination and Carbamylation Assisted by Peptide Retention Time Prediction.

The chromatographic behavior of peptides carrying citrulline and homocitrulline residues in proteomic two-dimensional (2D) liquid chromatography-mass spectrometry (LC-MS) experiments has been investigated. The primary goal of this study was to determine the chromatographic conditions that allow differentiating between arginine citrullination and deamidation of asparagine based on retention data, improving the confidence of MS-based identifications. Carbamylation was used as a reference point due to a high degree of similarity between modification products and anticipated changes in chromatographic behavior. We applied 2D LC-MS/MS (a high-pH-low-pH reversed phase (RP), hydrophilic interaction liquid chromatography (HILIC)-low-pH RP, and strong cation exchange (SCX)-low-pH RP) to acquire retention data for modified-nonmodified peptide pairs in the four separation modes. Modifications of a standard protein mixture were induced enzymatically (PAD-2) or chemically (urea) for citrullination and carbamylation, respectively. Deamidation occurs spontaneously. Similar retention shifts were observed for all three modifications in a high-pH RP (decrease) and a low-pH RP (increase), thus limiting the applicability of this 2D LC combination. HILIC on bare silica and strong cation exchange separations have been probed to amplify the effect of charge loss upon citrullination, with SCX demonstrating the most differentiating power: the elimination of basic residues upon citrullination/carbamylation results in an ∼58 mM KCl retention decrease, while retention of deamidated products decreases slightly.

Middle-up characterization of monoclonal antibodies by online reduction liquid chromatography-mass spectrometry

This study describes the fully automated middle-up characterization of monoclonal antibodies (mAbs) and next-generation variants by online reduction liquid chromatography-mass spectrometry (LC-MS). Proteins were trapped on-column and subjected to online desalting, denaturation and reduction prior to reversed phase elution of the created subunits in the MS. The evaluation of more than 20 different therapeutic proteins including full length mAbs (subclasses IgG1, IgG2 and IgG4), bispecific antibodies, antibody fragments, fusion proteins and antibody-drug conjugates (ADC) revealed that the online reduction method is as powerful as the widely applied offline sample preparation with dithiothreitol (DTT) as reducing agent and guanidine hydrochloride (Gnd.HCl) as denaturant and tackles some major disadvantages associated with the latter method, i.e. corrosion of stainless steel components, adduct formation impacting spectral quality and sample stability. The value of the online reduction LC-MS method is also enforced by its ability to reveal unstable antibody variants such as succinimide intermediates of asparagine deamidation and aspartic acid isomerization which are often lost when using the offline sample preparation method. The performance of the online reduction LC-MS set-up was verified and it was revealed that the method is precise with RSD values below 0.25% and 3.0% for retention time and area, respectively. Carry-over is within acceptable limits (< 0.5%) and the reducing buffer is stable up to 24 hours.

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.

Computational analysis of nonenzymatic deamidation of asparagine residues catalysed by acetic acid

In peptides and proteins, nonenzymatic deamidation of asparagine (Asn) residues can trigger some age-related diseases by disrupting the conformation of the biological proteins. In addition, Asn-residue deamidation, which has been observed in various protein preparations, is an important determinant of the quality of protein preparations. In the present study, we investigated the molecular mechanisms of Asn-residue deamidation catalysed by acetic acid, which is frequently used as a buffer in protein preparations. The calculations were conducted using an Asn residue capped with acetyl and methylamino groups on the N- and C-termini, respectively. Energy minima and transition-state geometries were optimised using B3LYP density functional theory (DFT) calculations. The relative energies of all optimised geometries obtained by the MP2 single-point energy calculations were corrected for zero-point energies calculated using the B3LYP DFT method. Asn-residue deamidation was divided roughly into two processes (cyclisation and deammoniation). Computational results indicate that cyclisation is rate-determining. A catalytic acetic acid molecule acted as a proton-transfer mediator in both processes. These results provide useful information for improving formulations of protein preparation.

Mechanisms of Deamidation of Asparagine Residues and Effects of Main-Chain Conformation on Activation Energy.

Deamidation of asparagine (Asn) residues is a nonenzymatic post-translational modification of proteins. Asn deamidation is associated with pathogenesis of age-related diseases and hypofunction of monoclonal antibodies. Deamidation rate is known to be affected by the residue following Asn on the carboxyl side and by secondary structure. Information about main-chain conformation of Asn residues is necessary to accurately predict deamidation rate. In this study, the effect of main-chain conformation of Asn residues on deamidation rate was computationally investigated using molecular dynamics (MD) simulations and quantum chemical calculations. The results of MD simulations for γS-crystallin suggested that frequently deamidated Asn residues have common main-chain conformations on the N-terminal side. Based on the simulated structure, initial structures for the quantum chemical calculations were constructed and optimized geometries were obtained using the B3LYP density functional method. Structures that were frequently deamidated had a lower activation energy barrier than that of the little deamidated structure. We also showed that dihydrogen phosphate and bicarbonate ions are important catalysts for deamidation of Asn residues.

NGR-TNF Engineering with an N-Terminal Serine Reduces Degradation and Post-Translational Modifications and Improves Its Tumor-Targeting Activity.

The therapeutic index of cytokines in cancer therapy can be increased by targeting strategies based on protein engineering with peptides containing the CNGRC (NGR) motif, a ligand that recognizes CD13-positive tumor vessels. We show here that the targeting domain of recombinant CNGRC-cytokine fusion proteins, such as NGR-TNF (a CNGRC-tumor necrosis factor-α (TNF) conjugate used in clinical studies) and NGR-EMAP-II, undergoes various post-translational modification and degradation reactions that lead to the formation of markedly heterogeneous products. These modifications include N-terminal cysteine acetylation or the formation of various asparagine degradation products, the latter owing to intramolecular interactions of the cysteine α-amino group with asparagine and/or its succinimide derivative. Blocking the cysteine α-amino group with a serine (SCNGRC) reduced both post-translational and degradation reactions. Furthermore, the serine residue reduced the asparagine deamidation rate to isoaspartate (another degradation product) and improved the affinity of NGR for CD13. Accordingly, genetic engineering of NGR-TNF with the N-terminal serine produced a more stable and homogeneous drug (called S-NGR-TNF) with improved antitumor activity in tumor-bearing mice, either when used alone or in combination with chemotherapy. In conclusion, the targeting domain of NGR-cytokine conjugates can undergo various untoward modification and degradation reactions, which can be markedly reduced by fusing a serine to the N-terminus. The SCNGRC peptide may represent a ligand for cytokine delivery to tumors more robust than conventional CNGRC. The S-NGR-TNF conjugate (more stable, homogeneous, and active than NGR-TNF) could be rapidly developed for clinical trials.

Mass spectrometric analysis of protein deamidation - A focus on top-down and middle-down mass spectrometry.

Deamidation of asparagine and glutamine alters protein structures and affects the chemical and biological properties of proteins. Protein deamidation has been demonstrated to be associated with protein folding, enzymatic activity, and degradation, as well as aging, cancer, and neurodegenerative diseases. To gain a better understanding on the biological roles of protein deamidation in aging and diseases, mass spectrometry (MS) has been employed in the identification of deamidated protein species and comprehensive characterization of deamidation sites. Three main MS approaches, top-down, middle-down, and bottom-up have been applied in the study of protein deamidation with high sensitivity, throughput, and accuracy. In this review, we discuss the application of top-down and middle-down MS in the study of protein deamidation, including sample preparation methods, separation strategies, MS and MS/MS techniques and data analysis. The advantages and drawbacks of these two approaches are also discussed and compared with those of the bottom-up method. The development of top-down and middle-down MS methods provides new strategies for protein deamidation analysis and gives new insights into the biological significance of protein deamidation in diseases.

Immunopeptidomic Analysis Reveals That Deamidated HLA-bound Peptides Arise Predominantly from Deglycosylated Precursors

The presentation of post-translationally modified (PTM) peptides by cell surface HLA molecules has the potential to increase the diversity of targets for surveilling T cells. Although immunopeptidomics studies routinely identify thousands of HLA-bound peptides from cell lines and tissue samples, in-depth analyses of the proportion and nature of peptides bearing one or more PTMs remains challenging. Here we have analyzed HLA-bound peptides from a variety of allotypes and assessed the distribution of mass spectrometry-detected PTMs, finding deamidation of asparagine or glutamine to be highly prevalent. Given that asparagine deamidation may arise either spontaneously or through enzymatic reaction, we assessed allele-specific and global motifs flanking the modified residues. Notably, we found that the N-linked glycosylation motif NX(S/T) was highly abundant across asparagine-deamidated HLA-bound peptides. This finding, demonstrated previously for a handful of deamidated T cell epitopes, implicates a more global role for the retrograde transport of nascently N-glycosylated polypeptides from the ER and their subsequent degradation within the cytosol to form HLA-ligand precursors. Chemical inhibition of Peptide:N-Glycanase (PNGase), the endoglycosidase responsible for the removal of glycans from misfolded and retrotranslocated glycoproteins, greatly reduced presentation of this subset of deamidated HLA-bound peptides. Importantly, there was no impact of PNGase inhibition on peptides not containing a consensus NX(S/T) motif. This indicates that a large proportion of HLA-I bound asparagine deamidated peptides are generated from formerly glycosylated proteins that have undergone deglycosylation via the ER-associated protein degradation (ERAD) pathway. The information herein will help train deamidation prediction models for HLA-peptide repertoires and aid in the design of novel T cell therapeutic targets derived from glycoprotein antigens.

Deamidation in Moxetumomab Pasudotox Leading to Conformational Change and Immunotoxin Activity Loss

Asparagine (Asn) deamidation is a common posttranslational modification in which Asn is converted to aspartic acid or isoaspartic acid. By introducing a negative charge, deamidation could potentially impact the binding interface and biological activities of protein therapeutics. We identified a deamidation variant in moxetumomab pasudotox, an immunotoxin Fv fusion protein drug derived from a 38-kDa truncated Pseudomonas exotoxin A (PE38) for the treatment of hairy-cell leukemia. Although the deamidation site, Asn-358, was outside of the binding interface, the modification had a significant impact on the biological activity of moxetumomab pasudotox. Surprisingly, the variant eluted earlier than its unmodified form on anion exchange chromatography, which often leads to the conclusion that it has a higher positive charge. Here we describe the characterization of the deamidation variant with differential scanning calorimetry and hydrogen-deuterium exchange mass spectrometry, which revealed that the Asn-358 deamidation caused the conformational changes in the catalytic domain of the PE38 region. These results provide an explanation for why the deamidation affected the biological activity of moxetumomab pasudotox and suggest the approach that can be used for process control to ensure product quality and process consistency.

Profiling the ‘deamidome’ of complex biosamples using mixed-mode chromatography-coupled tandem mass spectrometry

Deamidation is a spontaneous degenerative protein modification (DPM) that disrupts the structure and function of both endogenous proteins and various therapeutic agents. While deamidation has long been recognized as a critical event in human aging and multiple degenerative diseases, research progress in this field has been restricted by the technical challenges associated with studying this DPM in complex biological samples. Asparagine (Asn) deamidation generates L-aspartic acid (L-Asp), D-aspartic acid (D-Asp), L-isoaspartic acid (L-isoAsp) or D-isoaspartic acid (D-isoAsp) residues at the same position of Asn in the affected protein, but each of these amino acids displays similar hydrophobicity and cannot be effectively separated by reverse phase liquid chromatography. The Asp and isoAsp isoforms are also difficult to resolve using mass spectrometry since they have the same mass and fragmentation pattern in MS/MS. Moreover, the 13C peaks of the amidated peptide are often misassigned as monoisotopic peaks of the corresponding deamidated peptides in protein database searches. Furthermore, typical protein isolation and proteomic sample preparation methods induce artificial deamidation that cannot be distinguished from the physiological forms. To better understand the role of deamidation in biological aging and degenerative pathologies, new technologies are now being developed to address these analytical challenges, including mixed mode electrostatic-interaction modified hydrophilic interaction liquid chromatography (emHILIC). When coupled to high resolution, high accuracy tandem mass spectrometry this technology enables unprecedented, proteome-wide study of the 'deamidome' of complex samples. The current article therefore reviews recent advances in sample preparation methods, emHILIC-MS/MS technology, and MS instrumentation / data processing approaches to achieving accurate and reliable characterization of protein deamidation in complex biological and clinical samples.

Characterization of the acidic species of a monoclonal antibody using free flow electrophoresis fractionation and mass spectrometry

Antibody charge heterogeneity is one of the major product-related variants in recombinant biopharmaceuticals, which has been commonly monitored by imaged capillary isoelectric focusing (icIEF). Due to the challenges with sample recovery and fractionation, other charge-based analytical approaches have been explored as complementary methods allowing for further detailed charge variant characterization. This study describes the utilization of free flow electrophoresis (FFE) fractionation in combination with other analytical techniques, such as mass spectrometry for monoclonal antibody acidic variants characterization. The preparative FFE technique allowed for continuous sample separation and fluid phase fractionation of antibody charge isoforms. The monoclonal antibody starting material was fractionated by FFE, followed by purification and characterization. icIEF analysis demonstrated the purity of the fractions and comparability of the charge profiles between these two techniques. The intact molecular mass analysis revealed that glycation modification was highly enriched in the acidic fractions. SEC UV/Fluorescence method was developed to assess the levels of aggregation and fluorescent advanced glycation end-products (AGEs). Detailed peptide map was performed and revealed that acidic fractions were enriched in AGEs, methionine, tryptophan, histidine oxidation, asparagine deamidation, lysine glycation, carboxymethyl lysine, glycine to aspartic acid substitution compared to the main peak and starting material. The results indicate that acidic variants can account for a variety of low-level modifications present as very heterogeneous forms.