Why combining MALDI-MS and PEAs became the perfect assay for “me”

Assay choice is critical for any drug discovery program. Methyltransferase (MTase) enzymes are notoriously challenging due to the diversity of their substrates and that many substrates can achieve higher order methylation states, including Me1, Me2, and even Me3. Legacy assay technologies prefer to measure the metabolism of the SAM cofactor (the methyl donor), which leads to open questions around the achieved methylation state and impact of inhibitors. Developing antibodies to recognize distinct methylation states is challenging given the subtle differences of a single methyl group and the similarities of many substrate sequences. Traditional mass spectrometry can be often slow and restrictive. Our platform combining surface chemistry using polymeric enrichment arrays (PEAs) and state-of-the-art MALDI instrumentation overcomes these limitations with a label-free, direct, and ultra high-throughput readout of MTase activities. Download the case study focused on the protein arginine methyltransferase (PRMT5) enzyme and see how this assay platform is perfectly suited for confidently distinguishing “me” (methyl) groups and characterizing small molecule inhibitors to advance your drug discovery program. .

Measuring kinases and phosphatases using high-throughput MALDI and PEAs

Kinases and phosphatases are key enzymes in cellular signaling cascades, differentiation, and regulating protein function. Many kinases and phosphatases offer promising therapeutic strategies to combat various diseases. These enzymes act on various substrates, including proteins, nucleic acids, lipids, and small molecules, challenging the development of suitable and broadly applicable assay methodologies for measuring activity and characterizing inhibitors. Here, we demonstrate how combining innovative surface chemistry using polymeric enrichment arrays (PEAs) and state of the art MALDI mass spectrometry offers a label-free and high-throughput assay that is the gold standard for distinguishing distinct activities on virtually any substrate and quantitatively analyzing kinase and phosphatase inhibitors.

High-throughput assays for RNA polymerases using MALDI MS and PEAs

RNA polymerase enzymes are critical proteins involved in RNA stability and degradation and are implicated in regulating microRNAs, histone mRNAs, along with viral RNAs. Some RNA polymerases are important to restore degradation of oncogenic RNAs, offering a promising therapeutic strategy. Traditional biochemical assays that measure RNA activity rely on indirect measurements such as the generation of a pyrophosphate biproduct, or FRET assays that utilize a third component that binds to the newly synthesized strand to generate a FRET signal. Here, we demonstrate the use of MALDI mass spectrometry polymeric enrichment arrays (PEAs) to enable a direct readout of the RNA products, offering a robust and high-throughput assay to measure polymerase activity on any oligonucleotide.

High-throughput luminescence assays for ubiquitin ligases

Ubiquitin ligases (E3s) play a pivotal role in regulating protein stability and cellular signaling by catalyzing the transfer of ubiquitin to substrate proteins, marking them for degradation or functional modulation. Given their involvement in numerous diseases and targeted protein degradation modalities, E3 ligases have emerged as attractive therapeutic targets. Developing biochemical assays to measure E3 activity is essential for drug discovery and mechanistic studies. One accessible and robust approach involves luminescence-based assays that monitor ATP consumption. These assays not only provide a high-throughput means to measure E3 function, but also enable selectivity profiling across the broader family of ATP-dependent enzymes, including helicases and kinases, ensuring target specificity.

ASMS using MALDI MS and polymeric enrichment arrays (PEAs) for molecular glue discovery

The identification of small molecule binders to a distinct target is a common strategy to initiate drug discovery efforts, including for modalities that induce proximity, such as molecular glues. Affinity selection mass spectrometry, also known as ASMS, combined with polymeric enrichment arrays, offers unique benefits for identifying not only binders to virtually any target, including proteins and oligonucleotides such as RNA, but it also offers opportunities to detect proximity. Download this tech note to discovery how Peapod Bio’s innovative ASMS approach identified a small molecule binder and could also detect ternary complex formation.  

HTS for sortase A inhibitors using MALDI MS and polymeric enrichment arrays (PEAs)

The rise of antibiotic-resistant Staphylococcus aureaus necessitates new therapeutics to combat infections. Sortase A is an essential enzyme for virulence and a promising therapeutic target. Mass spectrometry combined with polymeric enrichment arrays (PEAs) offers a label-free and high-throughput technology to measure the protease and ligase activities in a multiplexed readout. Discover how Peapod Bio utilized this innovative approach to screen 200,000 compounds in days to reveal novel sortase A inhibitors.

Acetyltransferase assays using MALDI MS and polymeric enrichment arrays (PEAs)

Acetyltransferase enzymes are key players implicated in gene expression regulation, DNA replication and repair, where perturbation is associated with various diseases including cancer. Traditional assays rely on labels that lack selectivity or focus on cofactor metabolism, generating indirect measurements that can lead researchers down the wrong path. Learn how Peapod Bio’s innovative mass spectrometry assay offers the ability to directly measure substrate acetylation, including multiple acetylation events, providing clear, interpretable data faster to drive projects forward.