Differentiation of peptide isomers and epimers by radical-directed dissociation

Lambeth, Tyler R.; Julian, Ryan R.

doi:10.1016/bs.mie.2019.06.020

Cited by 9 publications

(12 citation statements)

References 42 publications

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“…(This value was measured in order to distinguish the tandem mass spectra of two isomeric peptides. It is determined by the ratios of the relative intensities of a pair of product ions that differ most between the isomeric pair and has been described in details by Julian .…”

Section: Resultsmentioning

confidence: 99%

“… a This R isomer value was obtained in order to distinguish the tandem mass spectra of two isomeric peptides (the iso Asp/Asp containing pair). It is determined by the ratios of the relative intensities of a pair of product ions that differ most between the isomeric pair as described by Julian . The value was calculated by dividing the ratio obtained for the iso Asp peptide by that determined for the Asp peptide. …”

Section: Resultsmentioning

confidence: 99%

“…Julian pioneered the use of radical-directed dissociation (RDD) in identifying amino acid isomers. The method has been successfully demonstrated in identifying iso Asp formation and epimerization of serine and other amino acids. , Smith reported excellent separation of four synthetic derivatives of β-amyloid peptides (residues 6–16) with Asp7 being unmodified, replaced with d -Asp, l - iso Asp, or d - iso Asp using SLIM (structures for lossless ion manipulations) ion mobility instrumentation. Elegant studies by Cooks demonstrated the use of ion chemistry to distinguish Asp- and iso Asp-containing peptides.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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.

View full text Add to dashboard Cite

show abstract

“…Indeed, the addition of fluorogenic residues on a substrate, including those previously reported for assaying FAAH activity (Ramarao et al, 2005), may alter its enzymatic conversion, decreasing assay sensitivity (Su et al, 2006;Ohira et al, 2018). Furthermore, isomerases are known to change molecular orientation of the substrate without changing its mass (Lambeth and Julian, 2019); however, changes in molecular orientation may lead to changes in solubility and, ultimately, changes in UPLC-MS/MS retention time. Therefore, the coupling of MS to the chromatographic step of liquid chromatography provides a sensitive method for determining enzyme activity with advantages over other methods described.…”

Section: Discussionmentioning

confidence: 99%

UPLC-MS/MS Method for Analysis of Endocannabinoid and Related Lipid Metabolism in Mouse Mucosal Tissue

et al. 2021

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The endocannabinoid system is expressed in cells throughout the body and controls a variety of physiological and pathophysiological functions. We describe robust and reproducible UPLC-MS/MS-based methods for analyzing metabolism of the endocannabinoids, 2-arachidonoyl-sn-glycerol and arachidonoyl ethanolamide, and related monoacylglycerols (MAGs) and fatty acid ethanolamides (FAEs), respectively, in mouse mucosal tissues (i.e., intestine and lung). These methods are optimized for analysis of activity of the MAG biosynthetic enzyme, diacylglycerol lipase (DGL), and MAG degradative enzymes, monoacylglycerol lipase (MGL) and alpha/beta hydrolase domain containing-6 (ABHD6). Moreover, we describe a novel UPLC-MS/MS-based method for analyzing activity of the FAE degradative enzyme, fatty acid amide hydrolase (FAAH), that does not require use of radioactive substrates. In addition, we describe in vivo pharmacological methods to inhibit MAG biosynthesis selectively in the mouse small-intestinal epithelium. These methods will be useful for profiling endocannabinoid metabolism in rodent mucosal tissues in health and disease.

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“…Certainly, RDD fragmentation was better than collision-induced dissociation (CID) in discriminating biologically-active peptides containing D-amino acids [ 99 ]. A comprehensive analysis protocol for differentiation of peptide isomers and epimers of lens crystallin using RDD was recently published [ 100 ]. Furthermore, the presence of the four Asp isomers in protein and peptides was identified using LC–MS, and three commercial enzymes without the need for synthesized reference peptides.…”

Section: Recent Advancement In Separation Techniques and The Impact On The Detections Of D-amino Acids And Daacpsmentioning

confidence: 99%

D-Amino Acids and D-Amino Acid-Containing Peptides: Potential Disease Biomarkers and Therapeutic Targets?

et al. 2021

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In nature, amino acids are found in two forms, L and D enantiomers, except for glycine which does not have a chiral center. The change of one form to the other will lead to a change in the primary structure of proteins and hence may affect the function and biological activity of proteins. Indeed, several D-amino acid-containing peptides (DAACPs) were isolated from patients with cataracts, Alzheimer’s and other diseases. Additionally, significant levels of free D-amino acids were found in several diseases, reflecting the disease conditions. Studying the molecular mechanisms of the DAACPs formation and the alteration in D-amino acids metabolism will certainly assist in understanding these diseases and finding new biomarkers and drug targets. In this review, the presence of DAACPs and free D-amino acids and their links with disease development and progress are summarized. Similarly, we highlight some recent advances in analytical techniques that led to improvement in the discovery and analysis of DAACPs and D-amino acids.

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Differentiation of peptide isomers and epimers by radical-directed dissociation

Cited by 9 publications

References 42 publications

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

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

UPLC-MS/MS Method for Analysis of Endocannabinoid and Related Lipid Metabolism in Mouse Mucosal Tissue

D-Amino Acids and D-Amino Acid-Containing Peptides: Potential Disease Biomarkers and Therapeutic Targets?

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