Isoaspartic Acid

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Zhaohui Sunny Zhou - One of the best experts on this subject based on the ideXlab platform.

  • mildly Acidic conditions eliminate deamidation artifact during proteolysis digestion with endoprotease glu c at ph 4 5
    Amino Acids, 2016
    Co-Authors: Kevin Ryan Moulton, Jared R Auclair, Zhaohui Sunny Zhou
    Abstract:

    Common yet often overlooked, deamidation of peptidyl asparagine (Asn or N) generates aspartic Acid (Asp or D) or Isoaspartic Acid (isoAsp or isoD). Being a spontaneous, non-enzymatic protein post-translational modification, deamidation artifact can be easily introduced during sample preparation, especially proteolysis where higher-order structures are removed. This artifact not only complicates the analysis of bona fide deamidation but also affects a wide range of chemical and enzymatic processes; for instance, the newly generated Asp and isoAsp residues may block or introduce new proteolytic sites, and also convert one Asn peptide into multiple species that affect quantification. While the neutral to mildly basic conditions for common proteolysis favor deamidation, mildly Acidic conditions markedly slow down the process. Unlike other commonly used endoproteases, Glu-C remains active under mildly Acid conditions. As such, as demonstrated herein, deamidation artifact during proteolysis was effectively eliminated by simply performing Glu-C digestion at pH 4.5 in ammonium acetate, a volatile buffer that is compatible with mass spectrometry. Moreover, nearly identical sequence specificity was observed at both pH’s (8.0 for ammonium bicarbonate), rendering Glu-C as effective at pH 4.5. In summary, this method is generally applicable for protein analysis as it requires minimal sample preparation and uses the readily available Glu-C protease.

  • Analysis of Isoaspartic Acid by Selective Proteolysis with Asp-N and Electron Transfer Dissociation Mass Spectrometry
    2015
    Co-Authors: Shujia Dai, Barry L Karger, Zhaohui Sunny Zhou
    Abstract:

    A ubiquitous yet underappreciated protein post-translational modification, Isoaspartic Acid (isoAsp, isoD or β-Asp), generated via the deamidation of asparagine or isomerization of aspartic Acid in proteins, plays a diverse and crucial role in ageing, as well as autoimmune, cancer, neurodegeneration and other diseases. In addition, formation of isoAsp is a major concern in protein pharmaceuticals, as it may lead to aggregation or activity loss. The scope and significance of isoAsp have, up to now, not been fully explored, as an unbiased screening of isoAsp at low abundance remains challenging. This difficulty is due to the subtle difference in the physicochemical properties between isoAsp and Asp, e.g., identical mass. In contrast, endoprotease Asp-N (EC 3.4.24.33) selectively cleaves aspartyl peptides but not the isoaspartyl counterparts. As a consequence, isoaspartyl peptides can be differentiated from those containing Asp and also enriched by Asp-N digestion. Subsequently, the existence and site of isoaspartate can be confirmed by electron transfer dissociation (ETD) mass spectrometry. As little as 0.5 % of isoAsp was detected in synthetic beta amyloid and cytochrome c peptides, even though both were initially assumed to be free of isoAsp. Taken together, our approach should expedite the unbiased discovery of isoAsp

  • protein isoaspartate methyltransferase mediated 18o labeling of Isoaspartic Acid for mass spectrometry analysis
    Analytical Chemistry, 2012
    Co-Authors: Min Liu, Janet Cheetham, Nina Cauchon, Judy Ostovic, Da Ren, Zhaohui Sunny Zhou
    Abstract:

    Arising from spontaneous aspartic Acid (Asp) isomerization or asparagine (Asn) deamidation, Isoaspartic Acid (isoAsp, isoD, or beta-Asp) is a ubiquitous nonenzymatic modification of proteins and peptides. Because there is no mass difference between isoaspartyl and aspartyl species, sensitive and specific detection of isoAsp, particularly in complex samples, remains challenging. Here we report a novel assay for Asp isomerization by isotopic labeling with 18O via a two-step process: the isoAsp peptide is first specifically methylated by protein isoaspartate methyltransferase (PIMT, EC 2.1.1.77) to the corresponding methyl ester, which is subsequently hydrolyzed in 18O-water to regenerate isoAsp. The specific replacement of 16O with 18O at isoAsp leads to a mass shift of 2 Da, which can be automatically and unambiguously recognized using standard mass spectrometry, such as collision-induced dissociation (CID), and data analysis algorithms. Detection and site identification of several isoAsp peptides in a mon...

  • Protein Isoaspartate Methyltransferase-Mediated 18O-Labeling of Isoaspartic Acid for Mass Spectrometry Analysis
    2012
    Co-Authors: Min Liu, Janet Cheetham, Nina Cauchon, Judy Ostovic, Da Ren, Zhaohui Sunny Zhou
    Abstract:

    Arising from spontaneous aspartic Acid (Asp) isomerization or asparagine (Asn) deamidation, Isoaspartic Acid (isoAsp, isoD, or beta-Asp) is a ubiquitous nonenzymatic modification of proteins and peptides. Because there is no mass difference between isoaspartyl and aspartyl species, sensitive and specific detection of isoAsp, particularly in complex samples, remains challenging. Here we report a novel assay for Asp isomerization by isotopic labeling with 18O via a two-step process: the isoAsp peptide is first specifically methylated by protein isoaspartate methyltransferase (PIMT, EC 2.1.1.77) to the corresponding methyl ester, which is subsequently hydrolyzed in 18O-water to regenerate isoAsp. The specific replacement of 16O with 18O at isoAsp leads to a mass shift of 2 Da, which can be automatically and unambiguously recognized using standard mass spectrometry, such as collision-induced dissociation (CID), and data analysis algorithms. Detection and site identification of several isoAsp peptides in a monoclonal antibody and the β-delta sleep-inducing peptide (DSIP) are demonstrated

  • analysis of Isoaspartic Acid by selective proteolysis with asp n and electron transfer dissociation mass spectrometry
    Analytical Chemistry, 2010
    Co-Authors: Shujia Dai, Barry L Karger, Zhaohui Sunny Zhou
    Abstract:

    A ubiquitous yet underappreciated protein post-translational modification, Isoaspartic Acid (isoAsp, isoD, or β-Asp), generated via the deamidation of asparagine or isomerization of aspartic Acid in proteins, plays a diverse and crucial role in aging, as well as autoimmune, cancer, neurodegeneration, and other diseases. In addition, formation of isoAsp is a major concern in protein pharmaceuticals, as it may lead to aggregation or activity loss. The scope and significance of isoAsp have, up to now, not been fully explored, as an unbiased screening of isoAsp at low abundance remains challenging. This difficulty is due to the subtle difference in the physicochemical properties between isoAsp and Asp, e.g., identical mass. In contrast, endoprotease Asp-N (EC 3.4.24.33) selectively cleaves aspartyl peptides but not the isoaspartyl counterparts. As a consequence, isoaspartyl peptides can be differentiated from those containing Asp and also enriched by Asp-N digestion. Subsequently, the existence and site of is...

Min Liu - One of the best experts on this subject based on the ideXlab platform.

Bernard Bodo - One of the best experts on this subject based on the ideXlab platform.

  • unprecedented occurrence of Isoaspartic Acid in a plant cyclopeptide
    Organic Letters, 2012
    Co-Authors: Damien Lacroix, Soizic Prado, Dennis Kamoga, John M Kasenene, Severine Zirah, Bernard Bodo
    Abstract:

    Three structurally related cycloheptapeptides, cyclocitropsides A–C, have been isolated from a MeOH extract of the root bark of Citropsis articulata, a medicinal plant in Uganda. Their sequences were elucidated on the basis of their MS/MS fragmentation, extensive 2D-NMR, chemical degradation, and biochemical modifications. Surprisingly, the sequence of cyclocitropside C differed from that of cyclocitropside B only by an Asp5/isoAsp5 substitution. This is the first report of an isoAsp residue in a plant cyclic peptide.

  • unprecedented occurrence of Isoaspartic Acid in a plant cyclopeptide
    Organic Letters, 2012
    Co-Authors: Damien Lacroix, Soizic Prado, Dennis Kamoga, John M Kasenene, Severine Zirah, Bernard Bodo
    Abstract:

    Three structurally related cycloheptapeptides, cyclocitropsides A–C, have been isolated from a MeOH extract of the root bark of Citropsis articulata, a medicinal plant in Uganda. Their sequences we...

Peter B Oconnor - One of the best experts on this subject based on the ideXlab platform.

  • differentiating n terminal aspartic and Isoaspartic Acid residues in peptides
    Analytical Chemistry, 2011
    Co-Authors: Nadezda P Sargaeva, Cheng Lin, Peter B Oconnor
    Abstract:

    Formation of Isoaspartic Acid (isoAsp) is a common modification of aspartic Acid (Asp) or asparagine (Asn) residue in proteins. Differentiation of isoAsp and Asp residues is a challenging task owin...

  • glutamine deamidation differentiation of glutamic Acid and γ glutamic Acid in peptides by electron capture dissociation
    Analytical Chemistry, 2010
    Co-Authors: Xiaojuan Li, Peter B Oconnor
    Abstract:

    Due to its much slower deamidation rate compared to that of asparagine (Asn), studies on glutamine (Gln) deamidation have been scarce, especially on the differentiation of its isomeric deamidation products: α- and γ-glutamic Acid (Glu). It has been shown previously that electron capture dissociation (ECD) can be used to generate diagnostic ions for the deamidation products of Asn: aspartic Acid (Asp) and Isoaspartic Acid (isoAsp). The current study explores the possibility of an extension of this ECD based method to the differentiation of the α- and γ-Glu residues, using three human Crystallin peptides (αA (1-11), βB2 (4-14), and γS (52-71)) and their potentially deamidated forms as model peptides. It was found that the z•-72 ions can be used to both identify the existence and locate the position of the γ-Glu residues. When the peptide contains a charge carrier near its N-terminus, the c+57 and c+59 ions may also be generated at the γ-Glu residue. It was unclear whether formation of these N-terminal diagn...

  • identification of aspartic and Isoaspartic Acid residues in amyloid β peptides including aβ1 42 using electron ion reactions
    Analytical Chemistry, 2009
    Co-Authors: Nadezda P Sargaeva, Cheng Lin, Peter B Oconnor
    Abstract:

    Amyloid β peptides are the major components of the vascular and plaque amyloid filaments in individuals with Alzheimer’s disease (AD). Although it is still unclear what initiates the disease, isomerization of aspartic Acid residues in Aβ peptides is directly related to the pathology of AD. The detection of isomerization products is analytically challenging, due to their similar chemical properties and identical molecular mass. Different methods have been applied to differentiate and quantify the isomers, including immunology, chromatography, and mass spectrometry. Typically, those methods require comparative analysis with the standard peptides and involve many sample preparation steps. To understand the role of Aβ isomerization in AD progression, a fast, simple, accurate, and reproducible method is necessary. In this work, electron capture dissociation (ECD) Fourier-transform ion cyclotron resonance mass spectrometry (FTICR MS) was applied to detect isomerization in Aβ peptides. ECD generated diagnostic f...

  • detecting deamidation products in proteins by electron capture dissociation
    Analytical Chemistry, 2006
    Co-Authors: Jason J Cournoyer, Peter B Oconnor
    Abstract:

    A nonenzymatic posttranslational modification of proteins and peptides is the spontaneous deamidation of asparaginyl residues via a succinimide intermediate to form a varying mixture of aspartyl and isoaspartyl residues. The isoaspartyl residue is generally difficult to detect particularly using mass spectrometry because Isoaspartic Acid is isomeric with aspartic Acid so that there is no mass difference. However, electron capture dissociation has demonstrated the ability to differentiate the two isoforms in synthetic peptides using unique diagnostic ions for each form; the cr• + 58 and zl-r − 57 fragment ions for the isoAsp form and the Asp side chain loss ((M + nH)(n-1)+• − 60) for the Asp form. Shown here are three examples of isoaspartyl detection in peptides from proteins; a deamidated tryptic peptide of cytochrome c, a tryptic peptide from unfolded and deamidated ribonuclease A, and a tryptic peptide from calmodulin deamidated in its native state. In all cases, the cr• + 58 and zl-r − 57 ions allowed...

  • deamidation differentiation of aspartyl from isoaspartyl products in peptides by electron capture dissociation
    Protein Science, 2005
    Co-Authors: Jason J Cournoyer, Jason L Pittman, Vera B Ivleva, Eric Fallows, Lucy Waskell, Catherine E Costello, Peter B Oconnor
    Abstract:

    Deamidation of asparaginyl and isomerization of aspartyl residues in proteins proceed through a succinimide intermediate producing a mixture of aspartyl and isoaspartyl residues. Isoaspartic Acid is an isomer of aspartic Acid with the Cβ incorporated into the backbone, thus increasing the length of the protein backbone by one methylene unit. This post-translation modification is suspected to contribute to the aging of proteins and to protein folding disorders such as Alzheimer’s disease, so that differentiating the two isomers becomes important. This manuscript reports that distinguishing aspartyl from isoaspartyl residues in peptides has been accomplished by electron capture dissociation (ECD) using a Fourier transform mass spectrometer (FTMS). Model peptides with aspartyl residues and their isoaspartyl analogs were examined and unique peaks corresponding to cn•+58 and zl−n-57 fragment ions (n, position of Asp; l, total number of amino Acids in the peptide) were found only in the spectra of the peptides with isoaspartyl residues. The proposed fragmentation mechanism involves cleavage of the Cα—Cβ backbone bond, therefore splitting the isoaspartyl residue between the two fragments. Also, a complementary feature observed specific to aspartyl residues was the neutral loss of the aspartic Acid side chain from the charge reduced species. CAD spectra of the peptides from the same instrument demonstrated the improved method because previously published CAD methods rely on the comparison to the spectra of standards with aspartyl residues. The potential use of the top-down approach to detect and resolve products from the deamidation of asparaginyl and isomerization of aspartyl residues is discussed.

Dirk Chelius - One of the best experts on this subject based on the ideXlab platform.

  • ion pair reversed phase high performance liquid chromatography method for the quantification of Isoaspartic Acid in a monoclonal antibody
    Journal of Chromatography B, 2014
    Co-Authors: Wolfram Kern, Robin Mende, Blandine Denefeld, Mirko Sackewitz, Dirk Chelius
    Abstract:

    Abstract Isomerization of aspartic Acid residues is one of the major causes of chemical degradation during the shelf life of biological pharmaceuticals. Monoclonal antibody biopharmaceuticals are typically stored at mildly Acidic pH conditions, which can lead to the isomerization reaction. The mechanism of this non-enzymatic chemical reaction has been studied in great detail. However, the identification and quantification of the isomerization sites in a given protein still remains a challenge. We developed an ion-pair reversed-phase HPLC method for the separation of an intact monoclonal antibody variant containing a single Isoaspartic Acid residue from its native counterpart. We identified and characterized the isomerization site using ion-pair reversed-phase HPLC mass spectrometry methods of the reduced and alkylated antibody and the enzymatically cleaved antibody. Lys-C followed by Asp-N digestion of the antibody was used for the identification of the isomerization site. Electron transfer dissociation (ETD) mass spectrometry was used to confirm the isomerization site at a DY motif at an aspartic Acid residue in the CDR-H3 region of the antibody. Tyrosine at the C-terminus of an aspartic Acid residue is typically not regarded as a hot spot for isomerization. Our findings suggest that it is not possible to predict isomerization sites in proteins with confidence and all aspartic Acid residues located in the CDR regions of antibodies must be considered as potential isomerization site due to the solvent exposure or the flexibility of these regions of the molecule. Additionally, the effect of the pH on the isomerization rate was evaluated using the ion-pair reversed-phase HPLC method, showing that at a lower pH the isomerization rate is faster. Storage at 25 °C for 6 months resulted in an increase of the amount of Isoaspartic Acid to 6.6% at pH 5.4, 6.0% at pH 5.8, and 5.6% at pH 6.2.

  • 18O labeling method for identification and quantification of succinimide in proteins.
    Analytical chemistry, 2007
    Co-Authors: Gang Xiao, Pavel V. Bondarenko, Jaby Jacob, Grace C. Chu, Dirk Chelius
    Abstract:

    We have developed a new method for identification and quantification of succinimide in proteins. The method utilizes 18O water to monitor succinimide hydrolysis. 18O-labeled Isoaspartic Acid and aspartic Acid peptides were produced by hydrolysis of a succinimide-containing protein in 18O water (H218O) followed by tryptic digestion in regular water (H216O). The peptides that had 18O incorporated were 2 Da heavier than their 16O native counterparts. The mass difference was detected and quantified by electrospray time-of-flight mass spectrometry. The amount of 18O incorporation into the Isoaspartic Acid- and aspartic Acid-containing peptides was used to quantify the amount of succinimide present in the native sample. The method was applied to analyze a degraded recombinant monoclonal antibody, which exhibited the accumulation of succinimide after storage in mildly Acidic buffers at elevated temperatures for a few weeks. We unambiguously identified amino Acid residue 30 located in the antibody light chain as ...

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