Analysis of Glutamine Deamidation: Products, Pathways, and Kinetics

Riggs, Dylan L.; Silzel, Jacob W; Lyon, Yana A.; Kang, Amrik S.; Julian, Ryan R.

doi:10.1021/acs.analchem.9b03127

Cited by 27 publications

(30 citation statements)

References 52 publications

(100 reference statements)

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“…Furthermore, CID is not very sensitive to structure in general. However, we have demonstrated previously that radical-directed dissociation (RDD) can identify covalently constrained stereoisomers on the basis of differences in fragmentation. , This approach can even successfully discriminate challenging isomers such as those derived from aspartic acid and glutamic acid. , RDD is initiated by photodissociation of a carbon–iodine bond, allowing for selective creation of a radical following activation with ultraviolet light. 3-Iodotyrosine was incorporated into our model peptide to facilitate RDD experiments.…”

Section: Resultsmentioning

confidence: 99%

“…41,42 can even successfully discriminate challenging isomers such as those derived from aspartic acid and glutamic acid. 43,44 RDD is initiated by photodissociation of a carbon−iodine bond, allowing for selective creation of a radical following activation with ultraviolet light. 3-Iodotyrosine was incorporated into our model peptide to facilitate RDD experiments.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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Probing the Stability of Proline Cis/Trans Isomers in the Gas Phase with Ultraviolet Photodissociation

Silzel

Murphree

Paranji

et al. 2020

J. Am. Soc. Mass Spectrom.

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Although most peptide bonds in proteins exist in the trans configuration, when cis peptide bonds do occur, they can have major impact on protein structure and function. The rapid identification of cis peptide bonds is therefore an important task. Peptide bonds containing proline are more likely to adopt the cis configuration because the ring connecting the side chain and backbone in proline flattens the energetic landscape relative to amino acids with free side chains. Examples of cis proline isomers have been identified in both solution and in the gas phase by a variety of structure-probing methods. Mass spectrometry is an attractive potential method for identifying cis proline due to its speed and sensitivity; however, the question remains of whether cis/ trans proline isomers originating in solution are preserved during ionization and manipulation within a mass spectrometer. Herein, we investigate the gas-phase stability of isolated solution-phase cis and trans proline isomers using a synthetic peptide sequence with a Tyr-Pro-Pro motif. A variety of dissociation methods were explored to evaluate their potential to distinguish cis/trans configuration, including collision-induced dissociation, radical-directed dissociation, and photodissociation. Only photodissociation employed in conjunction with extremely gentle electrospray and charge solvation by 18-crown-6 ether was able to distinguish cis/ trans isomers for our model peptide, suggesting that any thermal activation during transfer or while in the gas phase leads to isomer scrambling. Furthermore, the necessity for 18-crown-6 suggests that intramolecular charge solvation taking place during electrospray ionization can override cis/trans isomer homogeneity. Overall, the results suggest that solution-phase cis/trans proline isomers are fragile and easily lost during electrospray, requiring careful selection of instrument parameters and consideration of charge solvation to prevent cis/trans scrambling.

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Section: Resultsmentioning

confidence: 99%

Section: ■ Results and Discussionmentioning

confidence: 99%

Probing the Stability of Proline Cis/Trans Isomers in the Gas Phase with Ultraviolet Photodissociation

Silzel

Murphree

Paranji

et al. 2020

J. Am. Soc. Mass Spectrom.

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“…The current literature contains many studies on the characterization of such chemical modifications identified in both cellular proteins and biotherapeutics. − Examples of such chemical modifications include methionine and tryptophan oxidation, asparagine deamidation, as well as aspartate isomerization. − Glutamine deamidation is relatively rare and has been detected in long-lived proteins such as the crystallins. Characterization of this uncommon chemical modification has recently been reported by Julian . Most stable modifications display a very specific mass change such as +15.994 Da for tryptophan (Trp) and methionine (Met) oxidation and +0.984 Da for asparagine (Asn) deamidation and can be readily identified by liquid chromatography coupled to mass spectrometry (LC–MS).…”

Section: Introductionmentioning

confidence: 99%

“…Characterization of this uncommon chemical modification has recently been reported by Julian. 17 Most stable modifications display a very specific mass change such as +15.994 Da for tryptophan (Trp) and methionine (Met) oxidation and +0.984 Da for asparagine (Asn) deamidation 18 and can be readily identified by liquid chromatography coupled to mass spectrometry (LC−MS). Characterization of isoaspartate (isoAsp) formation in a protein by canonical MS can be a formidable task because the modified residue is isomeric with its unmodified counterpart.…”

Section: ■ Introductionmentioning

confidence: 99%

Unequivocal Identification of Aspartic Acid and isoAspartic Acid by MALDI-TOF/TOF: From Peptide Standards to a Therapeutic Antibody

Hui

Flick

Loo

et al. 2021

J. Am. Soc. Mass Spectrom.

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Aspartic acid (Asp) to isoaspartic acid (isoAsp) isomerization in therapeutic monoclonal antibodies (mAbs) and other biotherapeutics is a critical quality attribute (CQA) that requires careful control and monitoring during the drug discovery and production processes. The unwanted formation of isoAsp within biotherapeutics and resultant structural changes in the peptide backbone may negatively impact the efficacy, potency, and safety of the molecule or become immunogenic, especially if the isomerization occurs within the mAb complementarity determining region (CDR). Herein we describe a MALDI-TOF/TOF mass spectrometry method that affords unequivocal identification of the presence and the exact position of the isoAsp residue(s) in peptide standards ranging in size from a tripeptide to a docosapeptide (22 residues). In general, the peptide bond immediately N-terminal to the isoAsp residue is more susceptible to MALDI-TOF/TOF fragmentation than its unmodified counterpart. In some of the peptides evaluated in this study, fragmentation of the peptide bond C-terminal to the isoAsp residue (the aspartate effect) is also enhanced when compared to the control. Relative quantification by MALDI-TOF/TOF of this chemical modification is dependent upon a successful reversed-phase HPLC (rpHPLC) separation of the control and modified peptides. This method has also been validated on a therapeutic mAb that contains a well-documented isoAsp residue in the heavy chain CDR3 after forced degradation. Moreover, we also demonstrate that higher energy C-trap dissociation of only the singly charged species, and not the multiply charged form, of the isoAsp containing peptide, separated by rpHPLC, results in LC–MS/MS fragmentation that is highly consistent to that of MALDI-TOF/TOF.

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“…First, it has been reported that exposure to elevated pH (>10) increases the rate of the formation of succinimide or glutarimide intermediates due to the greater deprotonation of the peptide bond nitrogen at high pH values. The deamidation reaction proceeded more quickly at high pH, and ammonia was an effective general base catalyst for the deamidation of Asn and Gln residues in peptides [48,51]. The rate of deamidation was 6.5-fold faster in the solution which contained ammonia [48].…”

Section: Discussionmentioning

confidence: 99%

Comprehensive Evaluation of Different TiO2-Based Phosphopeptide Enrichment and Fractionation Methods for Phosphoproteomics

Wang

Yan

et al. 2022

Cells

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Protein phosphorylation is an essential post-translational modification that regulates multiple cellular processes. Due to their low stoichiometry and ionization efficiency, it is critical to efficiently enrich phosphopeptides for phosphoproteomics. Several phosphopeptide enrichment methods have been reported; however, few studies have comprehensively compared different TiO2-based phosphopeptide enrichment methods using complex proteomic samples. Here, we compared four TiO2-based phosphopeptide enrichment methods that used four non-phosphopeptide excluders (glutamic acid, lactic acid, glycolic acid, and DHB). We found that these four TiO2-based phosphopeptide enrichment methods had different enrichment specificities and that phosphopeptides enriched by the four methods had different physicochemical characteristics. More importantly, we discovered that phosphopeptides had a higher deamidation ratio than peptides from cell lysate and that phosphopeptides enriched using the glutamic acid method had a higher deamidation ratio than the other three methods. We then compared two phosphopeptide fractionation methods: ammonia- or TEA-based high pH reversed-phase (HpH-RP). We found that fewer phosphopeptides, especially multi-phosphorylated peptides, were identified using the ammonia-based method than using the TEA-based method. Therefore, the TEA-based HpH-RP fractionation method performed better than the ammonia method. In conclusion, we comprehensively evaluated different TiO2-based phosphopeptide enrichment and fractionation methods, providing a basis for selecting the proper protocols for comprehensive phosphoproteomics.

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Analysis of Glutamine Deamidation: Products, Pathways, and Kinetics

Cited by 27 publications

References 52 publications

Probing the Stability of Proline Cis/Trans Isomers in the Gas Phase with Ultraviolet Photodissociation

Probing the Stability of Proline Cis/Trans Isomers in the Gas Phase with Ultraviolet Photodissociation

Unequivocal Identification of Aspartic Acid and isoAspartic Acid by MALDI-TOF/TOF: From Peptide Standards to a Therapeutic Antibody

Comprehensive Evaluation of Different TiO2-Based Phosphopeptide Enrichment and Fractionation Methods for Phosphoproteomics

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