HPLC–UV–MS Analysis: A Source for Severe Oxidation Artifacts

Schweikart, Fritz; Hulthe, Gustaf

doi:10.1021/acs.analchem.8b05845

Cited by 12 publications

(7 citation statements)

References 16 publications

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“…Artificial oxidation can occur under normal operating conditions dependent on the field strength and the flow rate of the solution passing through the electrospray capillary 6 or as a result of gradual corrosion of stainless steel electrospray emitters 7 . Moreover, radicals formed by the light of UV detectors employed in online‐chromatography can generate unwanted artifact signals in the mass spectrum as well 8 …”

Section: What Is the Problem? Critical Observationsmentioning

confidence: 99%

Spectral quality overrides software score—A brief tutorial on the analysis of peptide fragmentation data for mass spectrometry laymen

König

2020

J Mass Spectrom

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The use of mass spectrometry has dramatically increased the research pace in the life sciences. The influence of the technique is enormous and its results can have far‐reaching consequences such as jail time when applied in forensics. Therefore, analytical chemists trained in proper procedure know that they must validate their experiments. However, those quality measures have not been adopted in a similar manner in the omics technologies even though the stakes are equally high. Reasons are, among others, the segregation of the data generation and data mining functions and an undue belief in software capabilities. In this article, problematic issues such as false or overinterpretation of data are discussed, and assistance is provided for mass spectrometry laymen to evaluate the quality of their results; a quick guide to mass spectral data interpretation of peptide fragmentation experiments, the basis of bottom‐up proteomics, is offered. Good science can only be generated in tight collaboration of principal investigator, analytical chemist, and bioinformatician so that the limits and the potential of each method and approach can be responsibly communicated.

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Section: What Is the Problem? Critical Observationsmentioning

confidence: 99%

Spectral quality overrides software score—A brief tutorial on the analysis of peptide fragmentation data for mass spectrometry laymen

König

2020

J Mass Spectrom

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“…In applications where the spectroscopic properties of the amino acid residues are applied, for example, detection in chromatography, UV wavelengths are used. During these kinds of exposure, the molecules may suffer from oxidation, misfolding, and aggregation, which can be detected as abundant artifacts in analysis or, in the case of light-stressed IgG1, cause immunogenic response. − Understanding the interaction between UV light and amino acid polymers can therefore help unravel the photoprocesses that occur to important proteins in vivo and increased understanding of the photochemical reactions can aid the design of more stable biopharmaceuticals. Predicting photostability and photoreactions in peptides and proteins remains challenging and requires more empirical studies in which powerful UV sources, such as femtosecond lasers, could be an advantage to use.…”

Section: Introductionmentioning

confidence: 99%

Characterization of Ultraviolet Photoreactions in Therapeutic Peptides by Femtosecond Laser Catalysis and Mass Spectrometry

et al. 2019

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Peptides and proteins have diverse ultraviolet (UV) photoreaction pathways that can be activated by the energy of the UV photons absorbed. Simple light sources such as lamps are conventionally used to study these photoreactions in solution. This work provides a proof of concept that femtosecond laser technology can function as a highly potent UV source in rapidly conducting UV photostability studies of peptides. Correspondingly, sufficient quantities of photoproducts were generated in 1 min or less, allowing for identification of known and new photomodifications in the therapeutic peptides somatostatin-14 and arginine vasopressin. Identical photoproducts were also generated with a conventional continuous source. The major modifications included N-formylkynurenine, a cross-link between Trp and Phe, a Tyr product with an NH3 loss, and disruption of an unstable disulfide bond into a complex mixture of a trisulfide bond and multiple scrambled dimeric products. In conclusion, femtosecond lasers are extremely useful to drive fast UV-induced reactions for high throughput screening of photostability and modifications in amino acid polymers.

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“…Third, significant oxidation of tryptophan, methionine and cysteine residues have been observed when passed through the UV diode array detectors (DAD) . It has been shown that the oxidation was mediated by hydroxyl free radicals produced by water molecules in the mobile phase upon UV (especially below 210 nm) irradiation in the DAD.…”

Section: Pitfalls Of Peptide Characterization Using Lc–msmentioning

confidence: 99%

Characterization of Synthetic Peptide Therapeutics Using Liquid Chromatography–Mass Spectrometry: Challenges, Solutions, Pitfalls, and Future Perspectives

Lian

Wang

Tian

et al. 2021

J. Am. Soc. Mass Spectrom.

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Synthetic peptides represent an important and expanding class of therapeutics. Despite having a relatively small size as compared to monoclonal antibodies and other proteins, synthetic peptides are subject to many complex structural modifications originating from the starting materials, manufacturing process, and storage conditions. Although mass spectrometry has been increasingly used to characterize impurities of synthetic peptides, systematic review of this field is scarce. In this paper, an overview of the impurities in synthetic peptide therapeutics is provided in the context of how the knowledge from detailed characterization of the impurities using liquid chromatography–mass spectrometry (LC–MS) can be used to develop the manufacturing process and control strategy for synthetic peptide therapeutics following the critical quality attribute (CQA)-driven and risk-based approach. The thresholds for identifying and controlling the impurities are discussed based on currently available regulatory guidance. Specific LC–MS techniques for identification of various types of impurities based on their structural characteristics are discussed with the focus on structural isomers and stereoisomers (i.e., peptide epimers). Absolute and relative quantitation methods for the peptide impurities are critiqued. Potential pitfalls in characterization of synthetic peptide therapeutics using LC–MS are discussed. Finally, a systematic LC–MS workflow for characterizing the impurities in synthetic peptide therapeutics is proposed, and future perspectives on applying emerging LC–MS techniques to address the remaining challenges in the development of synthetic peptide therapeutics are presented.

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HPLC–UV–MS Analysis: A Source for Severe Oxidation Artifacts

Cited by 12 publications

References 16 publications

Spectral quality overrides software score—A brief tutorial on the analysis of peptide fragmentation data for mass spectrometry laymen

Spectral quality overrides software score—A brief tutorial on the analysis of peptide fragmentation data for mass spectrometry laymen

Characterization of Ultraviolet Photoreactions in Therapeutic Peptides by Femtosecond Laser Catalysis and Mass Spectrometry

Characterization of Synthetic Peptide Therapeutics Using Liquid Chromatography–Mass Spectrometry: Challenges, Solutions, Pitfalls, and Future Perspectives

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