A tag-free collisionally induced fragmentation approach to detect drug-adducted proteins by mass spectrometry

Ballard, T. Eric; Dahal, Upendra P.; Bessire, Andrew J.; Schneider, Richard; Geoghegan, Kieran F.; Vaz, Alfin D. N.

doi:10.1002/rcm.7375

Cited by 11 publications

(8 citation statements)

References 27 publications

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“…Recent clinical progress demonstrated by various inhibitors including Neratinib, Afatinib, Ibrutinib, and VLX1570 has rekindled enthusiasm for covalent drugs. Similarly, data from the human genome project along with large-scale functional genomic studies have highlighted the need for selective probes which will enable biochemical analysis of enzyme function. , Mass spectrometry is well-suited to interrogate covalent protein modifications; in fact, several studies demonstrated the utility of mass spectrometry approaches to characterize small molecule–protein adducts, these include profiling of intact proteins , as well as top-down approaches to investigate antibody–drug conjugates, , metabolite-modified proteins, , and drug binding to serum albumin. , However, the potential for idiosyncratic toxicity resulting from drugs with a covalent mechanism-of-action continues to motivate the development of analytical platforms for rapid characterization of on- and off-target proteins. These assays would be implemented early in the drug discovery pipeline to assist in prioritization and triage of lead compounds.…”

Section: Discussionmentioning

confidence: 99%

Leveraging Gas-Phase Fragmentation Pathways for Improved Identification and Selective Detection of Targets Modified by Covalent Probes

Ficarro

Browne

Card³

et al. 2016

Anal. Chem.

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The recent approval of covalent inhibitors for multiple clinical indications has reignited enthusiasm for this class of drugs. As interest in covalent drugs has increased, so too has the need for analytical platforms that can leverage their mechanism-of-action to characterize modified protein targets. Here we describe novel gas phase dissociation pathways which yield predictable fragment ions during MS/MS of inhibitor-modified peptides. We find that these dissociation pathways are common to numerous cysteine-directed probes as well as the covalent drugs, Ibrutinib and Neratinib. We leverage the predictable nature of these fragment ions to improve the confidence of peptide sequence assignment in proteomic analyses and explore their potential use in selective mass spectrometry-based assays.

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Section: Discussionmentioning

confidence: 99%

Leveraging Gas-Phase Fragmentation Pathways for Improved Identification and Selective Detection of Targets Modified by Covalent Probes

Ficarro

Browne

Card³

et al. 2016

Anal. Chem.

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“…By use of a mass spectral technique previously reported by our lab, 34 we were able to specifically monitor the payloadcontaining protein peaks (DAR > 0) by MS as well as by SC. Briefly, both low and high collision energy (CE) scans were conducted over a wide mass range to accommodate coverage of the protein spectral envelope as well as small molecule payload fragment ions, and no isolation was performed in the quadrupole.…”

Section: Bioconjugate Chemistrymentioning

confidence: 99%

Determination of Antibody–Drug Conjugate Released Payload Species Using Directed in Vitro Assays and Mass Spectrometric Interrogation

et al. 2016

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Antibody-drug conjugates (ADC) are currently an active area of research, focused primarily on oncology therapeutics, but also to a limited extent on other areas such as infectious disease. The success of this type of targeted drug delivery is dependent upon many factors, one of which is the performance of the linker in releasing an active drug moiety under the appropriate conditions. As a tool in the development of linker/payload chemistry, we have developed an in vitro method for the identification of payload species released from ADCs in the presence of lysosomal enzymes. This method utilizes commercially available human liver S9 fraction as the source of these enzymes, and this has certain advantages over lysosomal fractions or purified enzymes. This article describes the characterization and performance of this assay with multiple ADCs composed of known and novel linkers and payloads. Additionally, we report the observation of incomplete degradation of mAb protein chains by lysosomal enzymes in vitro, believed to be the first report of this phenomenon involving an ADC therapeutic.

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“…Therefore, it was speculated that sutetinib could be covalently bound to HSA, similar to other TKIs. Based on the previously reported methods (Ballard et al, 2015), UPLC/Q‐TOF MS technology was used to study the differences in plasma stability between sutetinib and SNO.…”

Section: Resultsmentioning

confidence: 99%

Simultaneous determination of sutetinib and its active metabolite sutetinib N‐oxide in human plasma by liquid chromatography–tandem mass spectrometry: Evaluation of plasma stability

Qiu

Tang

et al. 2020

Biomedical Chromatography

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From the point of view of drug efficacy and safety, pharmacokinetic profiles of both In this work, a sensitive and reliable liquid chromatographic–tandem mass spectrometric method was established for simultaneous determination of sutetinib and N‐oxide metabolite (SNO) in human plasma and further applied to a pharmacokinetic study. Analytes were extracted from plasma samples (100 μl) via acetonitrile‐induced protein precipitation and separated on a C18 column using ammonium acetate with ammonium hydroxide and acetonitrile as the mobile phase. Positive electrospray ionization was carried out through multiple reaction monitoring with transitions of m/z 440.2 → 367.1 and 446.2 → 367.1 for sutetinib and SNO, respectively. The method was linear within the concentration range of 0.5–100 ng/ml for both analytes. The precision, accuracy, selectivity, recovery and matrix effect of this method all met the requirements of bioanalytical guidance. In addition, a plasma stability assessment demonstrated unexpected results. Sutetinib was prone to form covalent conjugates with plasma albumin in vitro. The degree of covalent binding increased with increasing temperature, resulting in a significant decrease in its plasma concentrations. However, SNO could not easily bind with albumin owing to steric hindrance or electronegativity. Furthermore, sutetinib and SNO remained stable when blood and plasma samples were kept on wet ice. The validated method was successfully employed for the pharmacokinetic evaluation of sutetinib in patients with advanced malignant solid tumors.

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A tag-free collisionally induced fragmentation approach to detect drug-adducted proteins by mass spectrometry

Cited by 11 publications

References 27 publications

Leveraging Gas-Phase Fragmentation Pathways for Improved Identification and Selective Detection of Targets Modified by Covalent Probes

Leveraging Gas-Phase Fragmentation Pathways for Improved Identification and Selective Detection of Targets Modified by Covalent Probes

Determination of Antibody–Drug Conjugate Released Payload Species Using Directed in Vitro Assays and Mass Spectrometric Interrogation

Simultaneous determination of sutetinib and its active metabolite sutetinib N‐oxide in human plasma by liquid chromatography–tandem mass spectrometry: Evaluation of plasma stability

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