Deamidation Rates Research Articles

Deamidation as a Consequence of β-Elimination of Phosphopeptides

Beta-elimination procedures often precede mass spectrometric analyses of phosphorylated peptides. Unfortunately, the commonly employed reaction conditions facilitate the deamidation of amide-containing residues. In addition to being 1 Da heavier than their amide counterparts, the newly created acidic residues greatly influence peptide tandem mass spectra. The effects of deamidation are investigated for five different amide-containing synthetic peptides exposed to beta-elimination conditions. MALDI-generated ions are analyzed with a tandem TOF mass analyzer. Methodologies for estimating the degree of deamidation from peptide mass spectra are presented, the influence that adjacent residues exert on the rate of deamidation is catalogued, and the impact that deamidation can have on peptide tandem mass spectra is demonstrated. The complications this side reaction can cause for automated data interpretation are also noted.

Asparagine Deamidation Perturbs Antigen Presentation on Class II Major Histocompatibility Complex Molecules

Post-translational protein modifications can be recognized by B and T lymphocytes and can potentially make "self"-proteins appear foreign to the immune system. Such modifications may directly affect major histocompatibility complex-restricted T cell recognition of processed peptides or may perturb the processing events that generate such peptides. Using the tetanus toxin C fragment protein as a test case, we show that spontaneous deamidation of asparagine residues interferes with processing by the enzyme asparagine endopeptidase (AEP) and contributes to diminished antigen presentation. Deamidation inhibits AEP action either directly, when asparagine residues targeted by AEP are modified, or indirectly, when adjacent Asn residues are deamidated. Thus, deamidation of long-lived self-proteins may qualitatively or quantitatively affect the spectrum of self-peptides displayed to T cells and may thereby contribute to the onset or exacerbation of autoimmune disease.

Effects of acidic N + 1 residues on asparagine deamidation rates in solution and in the solid state

The deamidation kinetics of four model peptides (AcGQNGG, AcGQNDG, AcGQNEG, and AcGQNQG) were studied in solution (70°C, pH 5–10) and in lyophilized solids [70°C, 50% relative humidity, “effective pH” (‘pH’) 5–10] containing polyvinyl pyrrolidone. AcGQNGG, AcGQNEG, and AcGQNQG degraded exclusively through Asn deamidation, whereas AcGQNDG also displayed Asp isomerization, and Asp‐Gly peptide bond cleavage. The pH/‘pH’‐rate profiles were consistent with a shift in the rate‐determining step of Asn deamidation from carbonyl addition to expulsion of ammonia with increasing pH. In solution, AcGQNGG deamidated up to 38‐fold faster than the other peptides, indicating the importance of steric effects of the N + 1 residue. AcGQNGG and AcGQNQG had up to 60 times slower rates of deamidation in the solid state than in solution. In contrast, the deamidation rates of AcGQNEG and AcGQNDG in the solid state were similar to those in solution. N + 1 Glu or Asp residue may enhance local hydration, so that the deamidation of Asn in the solid formulations actually proceeds in a solution‐like environment. © 2005 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:666–675, 2005

Asparaginyl deamidation in two glutamate dehydrogenase isoenzymes from Saccharomyces cerevisiae

The non-enzymatic deamidation of asparaginyl residues is a major source of spontaneous damage of several proteins under physiological conditions. In many cases, deamidation and isoaspartyl formation alters the biological activity or stability of the native polypeptide. Rates of deamidation of particular residues depend on many factors including protein structure and solvent exposure. Here, we investigated the spontaneous deamidation of the two NADP-glutamate dehydrogenase isoenzymes from Saccharomyces cerevisiae, which have different kinetic properties and are differentially expressed in this yeast. Our results show that Asn 54, present in Gdh3p but missing in the GDH1-encoded homologue, is readily deamidated in vitro under alkaline conditions. Relative to the native enzyme, deamidated Gdh3p shows reduced protein stability. The different deamidation rates of the two isoenzymes could explain to some extent, the relative in vivo instability of the allosteric Gdh3p enzyme, compared to that of Gdh1p. It is thus possible that spontaneous asparaginyl modification could play a role in the metabolic regulation of ammonium assimilation and glutamate biosynthesis.

Effects of Hydrophilic Cyclodextrins on Aggregation of Recombinant Human Growth Hormone

To evaluate the effect of hydrophilic cyclodextrins (CyDs) on the aggregation induced by different stresses and on the oxidation and deamidation of recombinant human growth hormone (rhGH). The aggregation of rhGH was induced by three denaturing techniques including chemical (4.5 M guanidine hydrochloride), thermal (differential scanning calorimetry), and interfacial denaturation (vortex agitation). The aggregates were characterized and quantified by UV spectrophotometry and size exclusion chromatography. The effects of hydrophilic CyDs on deamidation and oxidation rates of rhGH were studied by HPLC method. In both thermally and chemically induced aggregations, branched beta-CyDs significantly inhibited the aggregation of rhGH compared with the other alpha- and gamma-CyDs. This can explain that the beta-CyD cavity with branched sugar moieties may be relatively preferable in preventing the aggregation of rhGH. In contrast, 2-hydroxypropyl beta-CyD with surface activity was found to be effective in reducing the aggregation induced by interfacial denaturation compared with those of branched beta-CyDs. On the other hand, these hydrophilic CyDs showed no noticeable inhibitory effect on the oxidation and deamidation rates of rhGH. The results suggested that CyDs interact preferably with exposed hydrophobic side chains rather than aliphatic side chains of rhGH, resulting in the inhibition of aggregation but not the oxidation and deamidation rates. The different inhibitory effect of CyDs is dependent not only on the structure and property of CyD itself but also the nature of the denaturing stimulus. The current results suggested that hydrophilic beta-CyDs can effectively inhibit the aggregation of rhGH. Thus, hydrophilic beta-CyDs may be potentially useful excipients for parenteral preparation of rhGH.

Prediction of primary structure deamidation rates of asparaginyl and glutaminyl peptides through steric and catalytic effects.

The primary sequence dependence of deamidation has been quantitatively explained on the basis of a simple steric and catalytic model. Application to the known deamidation rates of peptides produces a table of coefficients that permits calculation of the known deamidation rates and prediction of deamidation rates for peptide sequences that have not yet been measured. This work permits a better understanding of deamidation, provides a prediction procedure for protein engineering, and facilitates improved computation of peptide and protein primary, secondary, tertiary, and quaternary structure deamidation rates.

Structure-dependent nonenzymatic deamidation of glutaminyl and asparaginyl pentapeptides.

Nonenzymatic deamidation rates for 52 glutaminyl and 52 asparaginyl pentapeptides in pH 7.4, 37.0 degrees C. 0.15 m Tris-HCl buffer have been determined by direct injection mass spectrometry. These and the previously reported 306 asparginyl rates have been combined in a self-consistent model for peptide deamidation. This model depends quantitatively upon peptide structure and involves succinimide, glutarimide and hydrolysis mechanisms. The experimental values and suitable interpolated values have been combined to provide deamidation rate values in pH 7.4, 37.0 degrees C. 0.15 m Tris-HCl buffer for the entire set of 648 single-amide permutations of ordinary amino acid residues in GlyXxxAsnYyyGly and GlyXxxGlnYyyGly. Thus, knowledge about sequence-dependent deamidation in peptides is extended to include very long deamidation half-times in the range of 2-50 years.

The estimation of glutaminyl deamidation and aspartyl cleavage rates in glucagon

The major hydrolytic degradation pathways of glucagon under acidic conditions are cleavage at Asp-9, Asp-15, and Asp-21, and deamidation at Gln-3, Gln-20, Gln-24, and Asn-28. The rate constants for these pathways were determined in the pH range 1–2.4 at 60 °C by kinetic data analysis of substrate and degradation product concentration–time profiles. Deamidation kinetics were determined using penta-peptide fragments of glucagon containing the labile amide residue. The accurate determination of the cleavage rate constants was confounded by the complexity of the degradation scheme of glucagon. Peptide cleavage kinetics were determined by degradation of glucagon and its cleavage fragments under identical conditions and the use of area-under-the-curve (AUC) and nonlinear regression methods of analysis. Glucagon degradation was first-order with respect to time and concentration in the range of 31–00 μM. Glutaminyl deamidation rate constants were first-order with respect to hydronium ion concentration and were similar for all three residues indicating a lack of sequence effects. The rate constants for Asp cleavage were not first-order with respect to hydronium ion concentration and cleavage at Asp-21 was slower than cleavage at Asp-9 and Asp-15 over the studied pH range.

Amide molecular clocks in drosophila proteins: potential regulators of aging and other processes

After synthesis and folding, peptides and proteins undergo changes in charge and conformation through nonenzymatic deamidation of asparaginyl and glutaminyl residues. Each amide has a specific deamidation rate that is genetically determined by the sequence of residues immediately adjacent in the peptide chain and by secondary, tertiary, and quaternary structure. By means of experimentally verified computations, we have determined the deamidation rates of 49 Drosophila peptides and proteins. These rates demonstrate that deamidation provides molecular clocks that are suitable for the regulation of Drosophila aging, development, and other biochemical processes. We have also determined the rates of deamidation for 17,886 other proteins from a wide variety of organisms. The distribution function of these deamidation rates demonstrates the suitability of amide residues as biomolecular clocks.

Deamidation of labile asparagine residues in the autoregulatory sequence of human phenylalanine hydroxylase

Two dimensional electrophoresis has revealed a microheterogeneity in the recombinant human phenylalanine hydroxylase (hPAH) protomer, that is the result of spontaneous nonenzymatic deamidations of labile asparagine (Asn) residues [Solstad, T. and Flatmark, T. (2000) Eur. J. Biochem.267, 6302-6310]. Using of a computer algorithm, the relative deamidation rates of all Asn residues in hPAH have been predicted, and we here verify that Asn32, followed by a glycine residue, as well as Asn28 and Asn30 in a loop region of the N-terminal autoregulatory sequence (residues 19-33) of wt-hPAH, are among the susceptible residues. First, on MALDI-TOF mass spectrometry of the 24 h expressed enzyme, the E. coli 28-residue peptide, L15-K42 (containing three Asn residues), was recovered with four monoisotopic mass numbers (i.e., m/z of 3106.455, 3107.470, 3108.474 and 3109.476, of decreasing intensity) that differed by 1 Da. Secondly, by reverse-phase chromatography, isoaspartyl (isoAsp) was demonstrated in this 28-residue peptide by its methylation by protein-l-isoaspartic acid O-methyltransferase (PIMT; EC 2.1.1.77). Thirdly, on incubation at pH 7.0 and 37 degrees C of the phosphorylated form (at Ser16) of this 28-residue peptide, a time-dependent mobility shift from tR approximately 34 min to approximately 31 min (i.e., to a more hydrophilic position) was observed on reverse-phase chromatography, and the recovery of the tR approximately 34 min species decreased with a biphasic time-course with t0.5-values of 1.9 and 6.2 days. The fastest rate is compatible with the rate determined for the sequence-controlled deamidation of Asn32 (in a pentapeptide without 3D structural interference), i.e., a deamidation half-time of approximately 1.5 days in 150 mm Tris/HCl, pH 7.0 at 37 degrees C. Asn32 is located in a cluster of three Asn residues (Asn28, Asn30 and Asn32) of a loop structure stabilized by a hydrogen-bond network. Deamidation of Asn32 introduces a negative charge and a partial beta-isomerization (isoAsp), which is predicted to result in a change in the backbone conformation of the loop structure and a repositioning of the autoregulatory sequence and thus affect its regulatory properties. The functional implications of this deamidation was further studied by site-directed mutagenesis, and the mutant form (Asn32-->Asp) revealed a 1.7-fold increase in the catalytic efficiency, an increased affinity and positive cooperativity of L-Phe binding as well as substrate inhibition.

The Relative Rates of Glutamine and Asparagine Deamidation in Glucagon Fragment 22–29 under Acidic Conditions

Our objective was to compare the relative rates of asparaginyl and glutaminyl deamidation in fragment 22–29 of the polypeptide hormone glucagon in acidic aqueous solutions. Reaction mixtures containing 22–29 (FVQWLMNT) or its degradation products were degraded at 60°C in dilute hydrochloric acid or phosphate buffer in the pH range 1–3. Degradation products were separated by high‐performance liquid chromatography and identified by amino acid sequencing, amino acid analysis, liquid chromatography‐mass spectrometry (LC‐MS), and matrix‐assisted laser desorption and ionization (MALDI). Nine major degradation products were identified, including asparaginyl and glutaminyl deamidated forms, aspartyl peptide cleavage of the asparaginyl deamidated products, and a cyclic imide intermediate. The pH dependences of rate constants for individual pathways were consistent with acid catalysis. Previous investigators have reported a greater susceptibility of asparagine residues to deamidation in neutral and alkaline solutions due to the formation of a more stable five‐membered succinimide intermediate. It has been suggested that asparagine may be more labile under acidic conditions also. We have observed a more facile deamidation for the glutamine residue under the acidic condition studied. It is proposed that the lower reactivity of the asparagine residue may be due to a decreased electrophilicity of its side chain carbonyl carbon imparted by a parallel cleavage pathway at this residue. © 2002 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:2332–2345, 2002

Effect of viscosity on the deamidation rate of a model Asn-hexapeptide.

The effect of viscosity on the deamidation rate of a model Asn-containing hexapeptide (l-Val-l-Tyr-Pro-l-Asn-Gly-l-Ala) was assessed in aqueous solution and in solids containing varying amounts of poly(vinyl pyrrolidone) (PVP) and water. Stability studies were conducted at 0.1 mg/mL peptide and 0-50% PVP (w/w) in aqueous solution, and at 5% (w/w) peptide and different relative humidities (31.6, 53.1, 74.4 and 96%) in the solid state. The parent peptide and its deamidation products were analysed by reverse-phase high-performance liquid chromatography. Deamidation rates decreased with increasing solvent viscosity in a manner described by a semi-empirical mathematical model developed to describe this relationship. The results suggest that the motion of the Asn side-chain along the reaction coordinate is a function of the macroscopic solvent viscosity. However, the apparent energy barrier for the diffusive movement of the side-chain appears to be less than the energy barrier for that associated with macroscopic viscosity. The dependence of the deamidation rate on viscosity in both viscous solution and hydrated solids further demonstrates the importance of mobility in peptide deamidation.

Protein deamidation.

A completely automatic computerized technique for the quantitative estimation of the deamidation rates of any protein for which the three-dimensional structure is known has been developed. Calculations of the specific deamidation rates of 170,014 asparaginyl residues in 13,335 proteins have been carried out. The calculated values have good quantitative reliability when compared with experimental measurements. These rates demonstrate that deamidation may be a biologically relevant phenomenon in a remarkably large percentage of proteins.

Insulin aspart (AspB28 human insulin) derivatives formed in pharmaceutical solutions.

To isolate and identify the main insulin aspart (AspB28 human insulin) derivatives formed in pharmaceuticals (pH 7.4 at 5 degrees C), to estimate rates of formation, and to determine their biologic potencies. Insulin aspart derivatives have been isolated by reversed-phase high-performance liquid chromatography (RP-HPLC), and identified by RP-HPLC, peptide mapping, amino acid analysis, mass spectrometry. and N-terminal amino acid sequence analysis. The main derivatives formed were isoAspB28, isoAspB3, AspB3, and desPheB1-N-oxalyl-ValB2 insulin aspart. At 5 degrees C, the rate constants were 0.00028/month for isoAspB28 and isoAspB3, 0.00024/month for AspB3, and 0.00013/month for desPheB1-N-oxalyl-ValB2 derivatives of insulin aspart. Unexpectedly, the rate of isomerization of B28 was high compared to the rate of B3 deamidation at both 5 degrees C and 45 degrees C. The N-terminal and especially the C-terminal of the B-chain are highly flexible, which may explain the high rate of isoAspB28 formation and that deamidation of AsnB3 occurs. All the derivatives had full in vivo biologic potencies. Except for isoAspB28 insulin aspart, the main derivatives formed in pharmaceuticals of insulin aspart and human insulin at pH 7.4 are similar. They are all fully active in vivo. In proteins, flexibility of the polypeptide chain seems more important than sequence in the formation of succinimides.

Controlling deamidation rates in a model peptide: Effects of temperature, peptide concentration, and additives

The rate of deamidation of the Asn residue in Val‐Tyr‐Pro‐Asn‐Gly‐Ala (VYPNGA), a model peptide, was determined at pH 9 (400 mM Tris buffer) as a function of temperature and peptide concentration. Over the temperature range 5–65°C, deamidation followed Arrhenius behavior, with an apparent activation energy of 13.3 kcal/mol. Furthermore, increasing the peptide concentration slows the rate of deamidation. Self‐stabilization with respect to deamidation has not been reported previously. The rate of deamidation was also determined in the presence of sucrose and poloxamer 407 (Pluronic F127). In both cases, the rate of deamidation was retarded by up to 40% at 35°C. In aqueous solutions containing poloxamer 407, the degree of stabilization is independent of formation of a reversible thermosetting gel. With sucrose, maximum reduction in the deamidation rate was attained with as little as 5% (w/v). Addition of sucrose results in a greater conformational preference for a type II β‐turn structure, which presumably is less prone to intramolecular cyclization and subsequent deamidation. © 2001 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:2141–2148, 2001

Action of protein-glutaminase on alpha-lactalbumin in the native and molten globule states.

The action of a novel protein-glutaminase from microorganisms on alpha-lactalbumin was investigated. When alpha-lactalbumin in the native state was incubated with protein-glutaminase, the deamidation proceeded gradually, i.e., the deamidation degree increased to 20% and 55% after 4 and 24 h, respectively. The transformation of alpha-lactalbumin from the native state to the molten globule state caused an increase in the rate of the enzyme-catalyzed deamidation, particularly in the early stage. The deamidation degree for the molten globule state reached 61% after 4 h, followed by a gradual increase to 66% after 24 h. CD spectral analyses of deamidated alpha-lactablumin revealed that the stability of the tertiary structure of alpha-lactablumin was closely related to the degree of deamidation, whereas the secondary structure was not affected by deamidation. Glutamine residues in alpha-lactalbumin to be modified by protein-glutaminase were identified as Gln[39], [43], [54], and [65]. Conformational characteristics of the amino acid sequence around these glutamine residues are discussed.

Deamidation of human proteins.

Deamidation of asparaginyl and glutaminyl residues causes time-dependent changes in charge and conformation of peptides and proteins. Quantitative and experimentally verified predictive calculations of the deamidation rates of 1,371 asparaginyl residues in a representative collection of 126 human proteins have been performed. These rates suggest that deamidation is a biologically relevant phenomenon in a remarkably large percentage of human proteins.

Stabilization of Recombinant Adenovirus: Site-Directed Mutagenesis of Key Asparagine Residues in the Hexon Protein

The quality and stability of recombinant adenovirus preparations for gene therapy trials are currently analyzed by anion-exchange HPLC. It was shown that the retention time of the adenovirus peak increased over the course of time upon storage in current liquid formulations. The number of isoaspartate residues at the outer surface of the particles also increased in parallel in these preparations. A recombinant adenovirus AV3760 was constructed with a modified hexon protein in which all four accessible Asn residues engaged in Asn–Gly pairs were changed into Leu (Asn244, Asn255, Asn437) or Ala (Asn276). The retention time of AV3760, as measured by HPLC, was unchanged after 2 months at +20°C as opposed to the control adenovirus. This demonstrates that the shift in retention time is caused by an increase in carboxyl groups on the outer surface of the virion due to deamidation of the above Asn residues. The modifications introduced in the hexon protein reduced the rate of Asn deamidation, without adverse effects on the infectivity of the virions. By reducing microheterogeneity in recombinant adenovirus, this work represents a significant advance in the development of a stable pharmaceutical vector for gene therapy.

Mass spectrometric evaluation of synthetic peptides as primary structure models for peptide and protein deamidation.

Solid-phase peptide synthesis and deamidation measurements using a novel mass spectrometric technique were carried out for 94 model asparaginyl peptides from 3 to 13 residues in length. Deamidation rates of these peptides in pH 7.4, 37.0 degrees C, 0.15 M Tris-HCl buffer were measured and evaluated. It was found that they validate the use of pentapeptide models as surrogates for the primary sequence dependence of peptide and protein deamidation rates and the discovery by difference of secondary, tertiary and quaternary structure effects. Deamidation of the pentapeptide models, compared with that of longer peptides of more intricate structure, is discussed, and the application of this technique to deamidation measurement of intact proteins is demonstrated.

Prediction of protein deamidation rates from primary and three-dimensional structure.

A method for the quantitative estimation of instability with respect to deamidation of the asparaginyl (Asn) residues in proteins is described. The procedure involves the observation of several simple aspects of the three-dimensional environment of each Asn residue in the protein and a calculation that includes these observations, the primary amino acid residue sequence, and the previously reported complete set of sequence-dependent rates of deamidation for Asn pentapeptides. This method is demonstrated and evaluated for 23 proteins in which 31 unstable and 167 stable Asn residues have been reported and for 7 unstable and 63 stable Asn residues that have been reported in 61 human hemoglobin variants. The relative importance of primary structure and three-dimensional structure in Asn deamidation is estimated.