Discovery and visualization of uncharacterized drug-protein adducts using mass spectrometry

Michael Riffle, Michael R. Hoopmann, Daniel Jaschob, Guo Zhong, Robert L. Moritz, Michael J. MacCoss, Trisha N. Davis, Nina Isoherranen, Alex Zelter

Preprint posted on 25 June 2021

Article now published in Analytical Chemistry at

It all adds up: using mass spectrometry to identify, understand, and characterise drug-protein adducts

Selected by Zhang-He Goh

Background of the preprint

Every day, we are exposed to many chemicals daily in the food we eat and the air we breathe. Often this exposure occurs in the form of pollution, but it can also arise when we take medication for the treatment of various diseases. These chemicals from extraneous sources are known as xenobiotics.

In our body, proteins fulfil important structural and functional roles. Unfortunately, they can react with these xenobiotics. The products that result from these reactions between xenobiotics and proteins—known as protein adducts—are responsible for various toxic effects. Sometimes these can manifest as allergies, which is why many medicines have adverse drug reactions. Evidently, modulating the formation of these adducts is important for human health: if we can modify their underlying mechanisms, we would be able to reduce the negative consequences of adduct formation, such as medicine side effects.

However, to date the identification and characterisation of these protein-chemical adducts remains a major challenge. Existing methods focus on chromatography, immunological techniques, and mass spectrometry to detect these adducts. However, these methods have two key limitations: they are not sufficiently sensitive, and often need to be built on a prior understanding of the chemical composition of these adducts, yet most adducts have not been characterised.

In this work, Riffle et al. describe the application of Magnum and Limelight, two new tools to study protein-drug adducts. Magnum is a customised adduct discovery algorithm and Limelight is a web-based analysis platform. Using CYP3A4, a metabolic enzyme responsible for oxidation, as well as raloxifene, the authors establish a workflow to show how Magum and Limelight can be used to characterise these adducts without the need for a prior understanding of the adducts formed.

Key findings of this preprint

(A) Magnum and Limelight

Riffle et al. developed Magnum, a ‘xenobiotic-protein adduct discovery algorithm’, to analyse tandem mass spectrometry (MS) (MS-MS) spectra. MS-MS is a highly sensitive and specific technique that offers significant advantages over single quadrupole MS. By involving multiple mass analysers, MS-MS is particularly well-suited to analyse biomolecules like proteins.

The authors highlight four features of Magnum that are key to its advantages compared to existing methods. First, Magnum analyses data pertaining to adduct-modified MS-MS ion series that allows better identification of adduct localisation, i.e. the site of adduct formation on the protein. Second, Magnum can restrict adduct localisation to specific amino acids, allowing chemical intuition to guide the search. Third, Magnum detects diagnostic reporter ions that are found in characteristic adduct fragmentation patterns. Fourth, Magnum differentiates between user-defined and undefined modifications. This feature reduces the chance of generating false positive identifications, which are more likely to occur during undefined searches.

Three criteria were used to assess Magnum: precision, recall, and accuracy. Precision was defined as the fraction of all answers that are correct; Magnum’s precision exceeded 0.9 when set at a 1% false discovery rate (set to minimise the rate of false positives generated by the algorithm). Furthermore, Magnum also exhibited a good recall, defined as the fraction of total correct answers identified, as well as a high accuracy of monoisotopic mass. Because many pharmaceutical drugs contain halogen isotopes like bromine and chlorine, the authors included code to enhance Magnum’s ability to correctly identify their respective adducts, a feature that is critical for their identification and subsequent characterisation.

To enable easier analysis of adduct data, Riffle et al. next developed Limelight, a web application to analyse, visualise, and share MS proteomics data.

(B) Identification of novel raloxifene adducts in CYP3A4 and P450-reductase

CYP3A4 is an enzyme that is responsible for the oxidation of many xenobiotics and requires P450-reductase to perform catalysis. CYP3A4 also oxidises raloxifene, producing several electrophilic species. A single 471 Da adduct on cysteine 239 has previously been identified.

Intriguingly, using Magnum and Limelight, Riffle et al. found 471 Da adducts not only on cysteine, but also on tryptophan and tyrosine, suggesting that raloxifene also reacts with these nucleophilic residues. Based on these data, the authors discuss the formation of adducts between raloxifene’s metabolites and CYP3A4, observations that may hint at key positions in the protein related to metabolite transport. These findings may in turn lead to important conclusions on the overall structure and function of CYP3A4 as well as its interactions with various ligands.

What I like about this preprint

I selected this preprint because of its potential impact on chemical biology, as well as its significance to related fields like medicine and toxicology (Figure 1). First, by identifying site-specific adducts based on MS spectra, this method may be extremely useful for proteomic applications, in which identifying adducts at selected sites have important downstream implications. For instance, the formation of these adducts could point to a particular residue’s reactivity, allowing for the development of methods to improve selectivity that would enable researchers to either selectively label (or avoid labelling) it. Alternatively, they may also shed light on the accessibility of the site. If the adduct is formed at a given amino acid residue, the amino acid residue could be in an important binding pocket, or perhaps in a surface-exposed environment. The upshot: a paradigm shift in how researchers identify druggable sites on proteins that could aid in drug development.

Figure 1. Significance of this work by Riffle et al.

Second, as Riffle et al. discuss in their study, understanding where adducts are formed could help to elucidate the structure and function of proteins. In their work, the authors cite substrate access and metabolite egress channels for this application. MS studies could help to provide evidence for hypotheses in cases where understanding the mechanism could be experimentally challenging.

Third, understanding the mechanism of formation of these adducts could enable improved drug design. While chemically reactive groups are well-understood in drug design, their metabolism in the body is less well defined. Indeed, the ability to understand and predict a drug’s metabolism, a significant contributor to drug toxicity, has been a major challenge in the field of pharmacokinetics for decades. While a few general mechanisms for adverse drug reactions arising from protein-xenobiotic adduct formation have been described, they are still mostly idiosyncratic. Thus, by profiling the reactivity of drugs’ metabolites with functionally important proteins in the body, this technology could enable the development of better medicines with fewer side effects.

Future directions

In addition to the scientific branches of innovation that I have highlighted above, I posit two other ways in which this interesting tool may move the field forward. The first pertains to the Limelight package itself: it is open source and has been made freely available on Github, and collaborations may be possible for further improvements that could be built on its existing capabilities. Given the interdisciplinary nature of research, in which scientists are increasingly encouraged to venture beyond their field of expertise, Limelight could be useful to a wider scientific audience. Researchers from different backgrounds, including computational chemists, chemical biologists, and medicinal chemists, could offer suggestions to improve Limelight’s capabilities. This would transform it into a more generalised platform for different applications.

Related to collaborations are considerations pertaining to usability, such as user design and user experience. These are important features of any application, even those developed for the scientific world. In this regard, researchers from different backgrounds could give feedback or even contribute towards Limelight’s design, as that will likely accelerate its use by the wider scientific community.


All images created using Microsoft Powerpoint and BioRender.

Questions for authors

  1. It is interesting that these methods are suitable for the characterisation of adducts formed from proteins, whether they react with metabolites or with inherently reactive xenobiotics, even without input pertaining to prior knowledge of adduct formation. How does Magnum differentiate between adduct formation at a given amino acid residue over another? In the CYP450 example provided in the preprint, for instance, how does Magnum identify adducts that have undergone addition at cysteine versus tryptophan or tyrosine, as they are likely to have the same mass?
  2. The adducts formed are likely to be stoichiometric, in that mono-adducts are formed between protein and metabolite in a 1:1 ratio, bi-adducts are formed between protein and metabolite in a 1:2 ratio, etc.. Given that MS-MS is a quantitative method for analysis, did you also observe these ratios between the reporter fragment ions (that arise from the adduct) and the other peptide fragments in the spectra?

Tags: mass spectrometry, protein adducts, proteomics

Posted on: 10 July 2021


Read preprint (1 votes)

Have your say

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

preLists in the biochemistry category:

Preprint Peer Review – Biochemistry Course at UFRJ, Brazil

Communication of scientific knowledge has changed dramatically in recent decades and the public perception of scientific discoveries depends on the peer review process of articles published in scientific journals. Preprints are key vehicles for the dissemination of scientific discoveries, but they are still not properly recognized by the scientific community since peer review is very limited. On the other hand, peer review is very heterogeneous and a fundamental aspect to improve it is to train young scientists on how to think critically and how to evaluate scientific knowledge in a professional way. Thus, this course aims to: i) train students on how to perform peer review of scientific manuscripts in a professional manner; ii) develop students' critical thinking; iii) contribute to the appreciation of preprints as important vehicles for the dissemination of scientific knowledge without restrictions; iv) contribute to the development of students' curricula, as their opinions will be published and indexed on the preLights platform. The evaluations will be based on qualitative analyses of the oral presentations of preprints in the field of biochemistry deposited in the bioRxiv server, of the critical reports written by the students, as well as of the participation of the students during the preprints discussions.


List by Marcus Oliveira

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.


List by Nadja Hümpfer et al.

20th “Genetics Workshops in Hungary”, Szeged (25th, September)

In this annual conference, Hungarian geneticists, biochemists and biotechnologists presented their works. Link:


List by Nándor Lipták


The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!


List by Osvaldo Contreras

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)


List by Madhuja Samaddar et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019


List by Dey Lab

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.


List by Pablo Ranea Robles


This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.


List by Sandra Franco Iborra

Also in the pharmacology and toxicology category:

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020


List by Ana Dorrego-Rivas

COVID-19 / SARS-CoV-2 preprints

List of important preprints dealing with the ongoing coronavirus outbreak. See for additional resources and timeline, and for full list of bioRxiv and medRxiv preprints on this topic


List by Dey Lab, Zhang-He Goh


Drug use in special populations

Any drugs that are being used in special populations: Patients with liver and kidney failure, in paediatrics, in geriatrics, and in pregnant or lactating patients. Includes the discovery of factors that could potentially affect drug use in these special populations.


List by Zhang-He Goh

Toxicology of toxicants, existing therapeutics, and investigational drugs

Preprints that describe the toxicology of environmental pollutants and existing and upcoming drugs. Includes both toxicokinetics and toxicodynamics, as well as technological improvements that will help in the characterisation of this field.


List by Zhang-He Goh

Antimicrobials: Discovery, clinical use, and development of resistance

Preprints that describe the discovery of new antimicrobials and any improvements made regarding their clinical use. Includes preprints that detail the factors affecting antimicrobial selection and the development of antimicrobial resistance.


List by Zhang-He Goh

Anticancer agents: Discovery and clinical use

Preprints that describe the discovery of anticancer agents and their clinical use. Includes both small molecules and macromolecules like biologics.


List by Zhang-He Goh

Advances in Drug Delivery

Advances in formulation technology or targeted delivery methods that describe or develop the distribution of small molecules or large macromolecules to specific parts of the body.


List by Zhang-He Goh