Mass Spectrometry–Driven Strategies for Membrane Protein–Targeted Drug Discovery

Membrane proteins play central roles in cellular signaling, transport, and communication and represent many clinically relevant drug targets, yet their low abundance and hydrophobicity make them difficult to study and exploit pharmacologically. Recent advances in mass spectrometry (MS) have transformed membrane protein–focused drug discovery by enabling high-throughput ligand screening, target identification, and binding-site deconvolution without the need for extensive protein purification. Affinity selection MS and emerging native MS approaches provide detailed, high-resolution insights into membrane protein–ligand interactions, proteoform specificity, and off-target effects directly from native-like membrane environments.

A recent review article published in Trends in Pharmacological Sciences (1) highlights evolving MS-based methodologies that are reshaping ligand discovery and mechanistic characterization for membrane protein drug targets. LCGC International spoke to Andrew Reiter and Dina Schuster, two of the authors of the article, about these methodologies.

What are the primary physiological functions of membrane proteins, and why are they considered attractive drug targets?

Membrane proteins have various functions such as signal transduction, transport of ions and nutrients, the catalysis of biochemical reactions, and intercellular communication and attachment. They occupy central roles in cellular communication and regulation, and they are often accessible from the extracellular environment, making them highly attractive drug targets.

Which classes of membrane proteins are most frequently targeted by FDA-approved drugs, and why?

More than 50% of all FDA-approved small-molecule drugs or biotherapeutics target integral membrane proteins, with roughly 30% targeting G protein–coupled receptors (GPCRs), followed by voltage-gated ion channels (8%), ligand-gated ion channels (7%), and transporters (7%). GPCRs represent the largest and most targeted family of membrane proteins because of their involvement in a wide range of signaling pathways that regulate numerous physiological and pathophysiological processes.

What challenges are associated with studying transmembrane proteins, particularly regarding their abundance and hydrophobicity?

Their low expression levels, and their hydrophobic transmembrane domains complicate expression, solubilization, purification, and structural characterization. Additionally, proteomics-based detection is further hindered by poor protease accessibility and the loss of membrane proteins during sample preparation.

How does mass spectrometry (MS) support drug discovery and development, especially for membrane protein targets?

MS is well-established for cytosolic protein target discovery, but it is limited for membrane protein applications. New MS-based strategies enable functional interrogation of membrane proteins for high-throughput compound screening, target identification, and binding site mapping across the membrane proteome.

Can you explain how affinity selection MS enhances high-throughput screening against membrane proteins?

Affinity selection MS (AS-MS) has been used to screen small-molecule ligands of various GPCRs and membrane proteins. However, because of constraints in membrane protein production yield and stability, these AS-MS screens were limited to relatively small libraries containing only 20 to 1500 compounds. To address the challenge of small library screens, researchers performed iterative rounds of affinity selection with a single pooled library approach. Their iterative screening method uses a series of selection rounds where associated compounds are eluted from the adenosine A2A receptor (A2AR) by chemical denaturation of the receptor and re-incubated with freshly prepared A2AR. This iterative approach enabled them to screen 20,000 compounds in one pool using both purified, stable A2AR and A2AR-embedded cell membranes.

What are the advantages of MS-based strategies that identify membrane protein drug targets without requiring protein purification?

Large-scale expression and purification of stable membrane proteins is difficult. Recently introduced workflows, such as Cys-Surf, cell surface TPP, LiP-Quant, and others, enable more comprehensive strategies to identify membrane protein drug targets in cell lysates or intact cells without requiring prior membrane protein purification.

How has native MS advanced the characterization of membrane proteins and their interactions with ligands?

Native MS analyses of membrane proteins typically require very pure preparations of membrane proteins in MS-compatible detergents. These detergents are then removed inside the mass spectrometer to allow for accurate mass measurements. Not only has MS instrumentation improved in recent years (enabling better removal of detergents), but researchers in the Robinson laboratory have also introduced workflows to analyze membrane proteins directly in their native environments, retaining annular lipids and endogenous interactors. This has allowed them to study protein–drug interactions in relevant contexts.

What unique insights can native MS provide into off-target effects and membrane proteoform-specific drug interactions?

Retaining the native surrounding of a membrane protein during native MS analyses enables the study of multiple proteins at once. Through incubations with a compound, mass shifts of all detected proteins can be measured, allowing for the detection of specific drug interactions. Native top-down MS, a workflow that combines native MS with fragmentation of the analyzed proteins, is a helpful method to detect proteoforms (distinct molecular forms of a protein, often a result of alternative splicing or post translational modifications) and their specific interactions in one experiment.

How can MS approaches help deconvolute complex cellular samples to identify ligandable binding sites on membrane proteins?

Activity-based protein profiling is a chemoproteomics method that quantifies the reactivity of specific amino acids toward reactive probes. In our review, we discuss two novel approaches: Cys-Surf (developed in the Backus laboratory) and Global Analysis of Surface Functionality (GASF; developed in the Lu laboratory). Cys-Surf first targets reactive cysteines and is then followed up with an enrichment of glycoproteins. In this way, the reactivity of cysteines can be probed directly on the cell surface. GASF uses a lysine-reactive probe to quantify the reactivity of cell-surface residing lysines.

In your opinion, what are the current limitations or challenges in applying MS to membrane protein–targeted drug discovery, and how might these be addressed?

The intrinsic properties of membrane proteins (high hydrophobicity, low abundance) make them challenging targets for MS-based drug discovery. Transmembrane domains are often poorly represented in traditional bottom-up proteomics experiments, limiting the sequence coverage and functional insights. In recent years, mainly through the development of selective enrichment methods, the targeting of cell surface proteins has become feasible. We believe that the development of organelle-specific enrichment methods and their combination with compound screens could be beneficial for the development of highly specific drugs with direct intracellular activity.

The throughput of native MS analyses is currently limited. If this could be increased and combined with large-scale drug screens, this could change the way we study protein–drug interactions.

References

1. Reiter, A. H.; Fehr, A.; Florea, R. et al. Mass Spectrometry-Based Strategies for Membrane Protein Pharmacology. Trends Pharmacol. Sci. 2025, S0165-6147 (25), 00236-6. DOI: 10.1016/j.tips.2025.10.012