Microreactor MS Screening Service for Enzyme and Reaction-Based Drug Discovery

Read the chemistry directly — no labels, no chromogenic substrates, no indirect proxies.

Microreactor mass spectrometry screening integrates miniaturised reaction vessels with direct MS detection, enabling researchers to monitor enzyme-catalysed reactions, measure substrate-to-product conversion, and identify inhibitors or activators in real time — from crude cell lysates, defined biochemical systems, or complex reaction mixtures — without the spectroscopic limitations that constrain fluorescence and absorbance-based assays.

At Creative Proteomics, our microreactor MS screening service is built for drug discovery teams who need to measure what actually happens in a reaction: the mass of the substrate consumed, the mass of the product formed, and how that ratio changes across a compound library. Whether the goal is enzyme inhibitor profiling, reaction mechanism confirmation, biocatalyst variant screening, or metabolic conversion monitoring, we deliver direct, label-free readouts at throughputs compatible with early-stage compound prioritisation. For broader high-throughput MS screening campaign design, our team can integrate microreactor workflows into a multi-assay strategy.

Key Advantages:

Direct product/substrate mass detection — no reporter, no chromogenic substrate, no fluorescent tag required.
Compatible with crude cell lysates, purified enzymes, and complex biological mixtures without extensive sample clean-up.
Semiquantitative-to-quantitative conversion measurement per well, enabling IC₅₀ estimation and rank-ordering of compound potency.
Applicable to enzyme classes without natural spectroscopic readouts — oxidoreductases, transferases, ligases, lyases, and isomerases.

Submit Your Microreactor MS Project View sample requirements

Microreactor MS screening service overview: miniaturised enzyme reaction wells feeding directly to DESI or ASAP mass spectrometry, delivering real-time substrate-to-product conversion data and inhibitor heatmap output for drug discovery.

What Is Microreactor MS Service Overview Tech Comparison Sample Demo Case Study FAQ

What Is Microreactor MS Screening?

Microreactor MS screening is an analytical approach in which enzymatic or chemical reactions are carried out in miniaturised volumes — typically in 96- or 384-well plate format, droplet microfluidic devices, or capillary flow-through chambers — and the reaction mixture is sampled directly by mass spectrometry without chromatographic separation. The mass spectrometer detects the molecular masses of substrates and products simultaneously, yielding a conversion measurement (substrate consumed / product formed) that is independent of the optical properties of the molecules involved.

The defining feature of the approach is the directness of the measurement. Conventional enzyme assays rely on a spectroscopic signal that must be coupled, directly or indirectly, to the chemical event of interest: a fluorescent substrate analogue, a chromogenic coupled enzyme, a bioluminescent ATP reporter. Each coupling step introduces assumptions, reagent dependencies, and interference susceptibilities that constrain which enzyme classes can be measured and in which biological matrices. Microreactor MS eliminates these intermediaries. If the substrate and product have distinct masses — and in the vast majority of biochemical reactions, they do — the conversion is directly observable.

This makes microreactor MS uniquely suited to enzyme classes that have historically resisted high-throughput screening: methyltransferases (where the methyl donor SAM and acceptor products lack chromophores), dehalogenases (where halogen substitution is not spectroscopically accessible without coupled assays), ammonia lyases (where stereochemical fidelity cannot be confirmed by absorbance), and a wide range of oxidoreductases and transferases where cofactor cycling assays introduce complexity and matrix interference. For dedicated enzyme activity and reaction mechanism studies requiring kinetic rate constants and Michaelis–Menten characterisation, our enzyme kinetics MS service provides a complementary deeper-mechanistic workflow. For continuous-flow microreactor configurations with residence-time control, our continuous-flow MS kinetics platform supports steady-state and pre-steady-state measurements.

Why Microreactor MS Changes the Enzyme Screening Equation

Screens enzymes that fluorescence assays cannot

The majority of transferases, isomerases, ligases, and lyases have no natural chromophore on their substrates or products. Coupled enzyme assays introduce additional variables and fail in complex biological matrices. Microreactor MS reads the reaction chemistry directly, making any enzyme with a substrate of known mass screenable without assay development investment.

Works in crude biological matrices

Cell lysates, microsomal fractions, and whole-cell reaction mixtures can be spotted directly to the MS inlet. The mass spectrometer discriminates substrate from product by accurate mass regardless of co-eluting cellular components — a capability that fluorescence and absorbance readers cannot match in optically complex samples.

Delivers identity, not just a signal proxy

A fluorescence signal tells you that something changed. Microreactor MS tells you what changed: the substrate's exact mass decreased, the product's exact mass appeared, and any unexpected metabolite or reactive intermediate can be identified by its m/z — providing hit confirmation and early liability flagging in the same experiment.

Scales to 96- and 384-well plate throughput

Ambient ionisation interfaces (DESI, ASAP, and open-port sampling probe) allow the MS to sample reaction wells at rates of one well per 10–60 seconds without LC separation, achieving practical throughputs of 1,500–8,000 data points per day — sufficient for focused inhibitor library screening and enzyme variant panel assessment.

Service Overview — Microreactor MS Screening Modes

We offer four operational modes within our microreactor MS screening service, each matched to a distinct drug discovery context. All modes share the same core MS detection principle — direct ambient ionisation coupled to high-resolution or triple-quadrupole MS — but differ in reaction format, throughput setting, and data output. Our scientists consult on assay design before project initiation to confirm substrate–product mass differentials, matrix compatibility, and the appropriate ionisation interface for each enzyme class.

MODE 1

Enzyme Inhibitor Panel Screening

A defined compound set (focused library, fragment library, or compound class) is tested against a single purified enzyme or enzyme-containing lysate. Each reaction well receives compound at a fixed concentration; conversion is measured by the substrate/product ratio at a fixed time point.

Output: per-compound conversion percentage, ranked inhibitor list, hit confirmation by product mass verification.
Format: 96-well or 384-well plate; duplicate or triplicate measurements per compound.
Throughput: up to 384 compounds per day in single-concentration mode; dose-response series available for hit follow-up.
Enzyme classes screened include methyltransferases, kinases (direct product detection), dehalogenases, aminotransferases, and lyases.

MODE 2

Enzyme Variant and Biocatalyst Activity Profiling

Multiple enzyme variants (wild-type plus point mutants, or a metagenomic enzyme panel) are screened against one or more substrates to identify variants with enhanced activity, altered selectivity, or expanded substrate scope.

Output: activity heatmap across enzyme × substrate grid; substrate scope ranking; variant prioritisation for directed evolution or compound optimisation.
Format: spotted-plate DESI-MS or open-port sampling probe; crude cell lysate compatible.
Applications: directed evolution support, chemoenzymatic route scouting, natural product biosynthetic enzyme characterisation. See also our natural product MS discovery service for downstream structural elucidation of enzymatic products.

MODE 3

Metabolic Conversion and Biotransformation Monitoring

Drug candidates or compound series are incubated with enzymatic systems (microsomes, S9 fractions, hepatocytes, recombinant CYPs) and the resulting metabolite landscape is profiled by direct MS detection — identifying primary metabolic products, reactive intermediate formation, and relative conversion rates without pre-extracted metabolite standards.

Output: metabolite identity by accurate mass, relative abundance at defined time points, conversion rate comparison across compound series.
Compatible with microsomal and S9 fractions; complementary to our metabolic stability LC-MS/MS service when quantitative half-life data is required.
Useful for early metabolic soft-spot identification and structural liability flagging within a compound series.

MODE 4

Covalent and Reactive Compound Reaction Profiling

Compounds designed or suspected to covalently modify an enzyme active site are profiled for reaction efficiency, selectivity, and product characterisation. Microreactor MS quantifies covalent adduct formation by accurate mass of the modified substrate or enzyme peptide, distinguishing productive engagement from non-selective reactivity.

Output: covalent adduct mass confirmation, reaction stoichiometry estimate, selectivity comparison across target and off-target substrates.
Complements our covalent fragment screening platform for warhead-level target engagement validation.
Applicable to electrophilic compound classes: acrylamides, chloroacetamides, cyanoacrylates, and Michael acceptors.

Analytical Workflow

Five stages from reaction design to ranked compound output:

Assay design and substrate–product mass verification

Before any compound screening begins, we verify that the enzyme reaction produces a detectable mass differential. The substrate and product masses (including any cofactor adducts, salt adducts, or in-source fragment ions) are mapped by direct infusion of authentic standards or confirmed reaction products. MS acquisition parameters (scan range, resolution, ionisation polarity, fragmentation if required) are optimised for the specific substrate–product pair. This step takes 1–2 days and defines the acceptance criteria for all screening data.

Reaction plate preparation and incubation

Enzyme reactions are set up in 96- or 384-well plates at defined substrate concentration, enzyme concentration, cofactor conditions (if required), and time point. Compound solutions are dispensed at the target screening concentration (typically 10–100 µM, adjusted for enzyme Km and expected inhibitor potency). DMSO concentrations are controlled to ≤1% throughout to avoid enzyme denaturation. Positive (full inhibition) and negative (DMSO-only) controls occupy defined well positions in each plate.

Direct ambient MS sampling

Completed reaction plates are positioned on the automated sampling stage of the DESI or open-port sampling probe interface. The MS source continuously samples from defined well positions — spraying solvent across each spot (DESI) or aspirating a defined volume (open-port) — at a sampling rate of approximately one well per 10–60 seconds. No chromatographic column is used; raw spectra are acquired at each well position and correlated to the plate map.

Conversion calculation and quality control

Raw spectra are processed to extract substrate and product ion abundances at the expected m/z values. Conversion percentage is calculated as [product / (product + substrate)] × 100 for each well. Plate-level QC metrics are applied: coefficient of variation across replicate control wells, Z-factor calculation for each plate, signal-to-noise check against background wells. Plates with Z-factor < 0.5 are flagged for re-measurement.

Hit identification and ranked output delivery

Compounds producing conversion values below the hit threshold (typically defined as mean control − 3 × SD, or ≥50% inhibition relative to DMSO vehicle) are designated as primary hits. Hit confirmation is performed by repeat measurement and, where appropriate, by MS2 fragmentation of the product ion to confirm molecular identity. Deliverables include a per-compound conversion table, ranked hit list with accurate-mass confirmation, plate-level QC report, and a heatmap visualisation of conversion across the screened library.

Microreactor MS screening workflow: reaction plate preparation, enzyme incubation with compound library, direct DESI or open-port MS sampling, conversion calculation and Z-factor QC, ranked hit list and heatmap delivery.

Applications by Drug Discovery Stage

Microreactor MS is most impactful where direct chemical identity of reaction products matters — not just a signal change, but a confirmed molecular transformation.

Inhibitor Identification for Spectroscopically Challenging Enzymes

Methyltransferases, halogenases, ammonia lyases, carbamoyltransferases, and other enzyme classes where coupled fluorescence assays are unreliable or unavailable are directly accessible by microreactor MS. The enzyme class does not determine screenability — the substrate mass difference does.

Output: Ranked inhibitor list with per-compound conversion; confirmation of hit identity by product accurate mass; selectivity profile across related enzyme family members if multiple enzyme variants are screened in parallel.

Directed Evolution and Biocatalyst Optimisation Support

Screening enzyme variant libraries for activity improvements requires high-throughput, label-free readouts from crude cell lysates where spectroscopic assays cannot function reliably. Microreactor MS converts raw well-plate data into an activity heatmap — identifying high-converting variants in a 96- or 384-well format without prior protein purification.

Output: Activity heatmap across variant × substrate grid; substrate scope determination; hit variants ranked by conversion for sequence-activity analysis. Particularly relevant to biocatalytic route development for drug substance manufacturing, enzyme engineering for pharmaceutical applications, and directed evolution of biosynthetic enzymes.

Chemoenzymatic Reaction Scouting

Novel synthetic routes in pharmaceutical chemistry increasingly incorporate enzymatic steps. Microreactor MS rapidly evaluates whether a candidate enzyme achieves the desired bond formation or transformation at each substrate in a series — establishing substrate scope before investing in preparative-scale reaction development.

Output: Go/no-go substrate compatibility table with product mass confirmation; relative conversion rates across substrate series; identification of undesired side-products by mass.

Cell-Based Metabolic Reaction Monitoring

Compounds that modulate intracellular enzyme activity leave a metabolic signature detectable by direct MS analysis of cell extracts. Microreactor MS applied to post-treatment cell lysate allows monitoring of specific enzymatic product formation — confirming target engagement at the biochemical level without requiring a cell-permeable fluorescent probe. This complements our cell-based MS drug screening platform for broader cellular drug response characterisation.

Output: Enzyme product concentration in cell lysate across compound concentrations; dose-response correlation between compound exposure and enzymatic readout; confirmation that cellular target engagement produces the expected biochemical consequence.

ABPP-Compatible Reactive Probe Profiling

Activity-based protein profiling probes that covalently label active enzymes can be paired with microreactor MS to confirm probe reactivity against recombinant enzyme targets — verifying probe-to-enzyme stoichiometry and identifying unreacted probe or probe-adduct mass shifts that inform probe optimisation. Detailed residue-level engagement studies can be extended through our high-throughput ABPP platform.

Output: Probe-labelled enzyme adduct mass; labelling efficiency as a function of probe concentration; selectivity fingerprint across a panel of related enzyme targets in a single experiment.

Natural Product Reaction Diversity Screening

Complex mixtures from natural product extracts, fermentation broths, or semi-synthetic libraries contain multiple reactive components. Microreactor MS can simultaneously monitor which mixture components react with an enzyme substrate — identifying bioactive constituents by their product-forming mass signatures without prior fractionation. For upstream isolation and dereplication workflows, our natural product MS discovery service supports extract characterisation before reaction screening.

Output: Product-forming mass signatures per mixture component; identification of active constituents by product m/z; guidance for bioassay-guided fractionation priorities.

Technology Comparison: Microreactor MS vs Alternative Enzyme Screening Platforms

Platform	Detection Principle	Label Required?	Crude Lysate Compatible?	Identity Confirmed?	Enzyme Class Limitation	Throughput (wells/day)
Microreactor MS / DESI-MS (this service)	Direct ambient ionisation → product mass detection	None	✅ Yes — full matrix tolerance	✅ Yes — by accurate mass ± MS2	None — any enzyme producing a mass-distinguishable product	384–8,000+
Fluorescence-Based Assay (FP, HTRF, fluorogenic substrate)	Fluorescence polarisation, FRET, or product fluorescence change	Required — fluorescent probe or labelled substrate	❌ Limited — autofluorescence, inner filter effects	❌ No — indirect signal proxy	High — enzymes producing chromophoric or fluorescent products	Up to 100,000+
RapidFire MS	Rapid solid-phase extraction → triple-quadrupole MS	None	⚠️ Partial — SPE matrix tolerance varies	✅ Yes — by MRM transition	Pre-defined MRM transitions required per analyte	Up to ~3,000
Acoustic Ejection MS (AEMS / ECHO-MS)	Acoustic droplet ejection → open-access ESI-MS	None	⚠️ Partial — requires low-salt conditions	✅ Yes — by accurate mass	Salt and matrix sensitivity limits biological complexity	Up to 10,000+
MALDI-TOF HTS	Matrix-assisted laser desorption → mass detection from spotted arrays	None	⚠️ Partial — matrix preparation required	✅ Yes — by mass	High molecular weight bias; co-crystallisation affects low-mass analytes	Up to 20,000+
Coupled Colorimetric Assay (e.g. NADH-linked)	Absorbance change via coupled enzyme reporter	Indirect — coupled enzyme system required	❌ No — cofactor depletion, competing reactions	❌ No — signal is indirect	Only enzymes with NADH/NADPH-linked coupled assay chemistry	Up to 1,536

For projects where RapidFire-format SPE-MS is preferred, our RapidFire MS service provides validated workflows. For acoustic ejection-based MS platforms with ultra-high throughput requirements, see our acoustic ejection MS service. For MALDI-based HTS applications, our MALDI-TOF HTS service covers spotted-array and off-line MALDI plate workflows.

Sample Requirements

Component	Format Options	Recommended Input	Minimum Input	Key Notes
Enzyme (purified)	Recombinant protein in assay-compatible buffer	50–200 µg total (scaled to assay volume)	10 µg (if Km and optimal conditions are known)	Provide enzyme activity data, Km, kcat if available; confirm no glycerol >5% or detergents above CMC in storage buffer; add protease inhibitor cocktail before shipping
Enzyme (crude lysate)	Cell lysate, microsomal fraction, S9 fraction, or whole-cell extract	≥ 0.5 mL at 1–5 mg/mL total protein	100 µL at ≥ 1 mg/mL	Specify expression system (E. coli, HEK293, Sf9, yeast); note any endogenous enzyme activities in the lysate that may confound substrate conversion; ship on dry ice
Substrate	Dissolved in DMSO (preferred) or aqueous buffer	10 mM stock solution; ≥ 100 µL	5 mM stock; 50 µL	Provide accurate molecular weight (monoisotopic) and known purity; if substrate is susceptible to non-enzymatic hydrolysis or oxidation, note stability data; do not supply as dry powder unless pre-arranged
Compound Library	Individual wells in 96- or 384-well plate (DMSO stocks)	10 mM in DMSO; ≥ 50 µL per well	2 mM in DMSO; 10 µL per well	Provide SDF or compound ID list with molecular weights; DMSO concentration in final assay must not exceed 1%; compounds with MS-interfering counterions (TFA > 0.05%) should be noted; aggregating compounds may require orthogonal validation
Cofactors / Cosubstrates	Aqueous solutions	10× concentrated stock; ≥ 200 µL	2× concentrated; 50 µL	SAM, NAD+, NADPH, ATP, CoA, PLP, and other cofactors must be fresh (< 4 weeks from preparation); indicate stability conditions; provide if not commercially sourceable by our team
Reference Inhibitors (positive controls)	10 mM DMSO stocks	≥ 50 µL	10 µL	At least one reference inhibitor with known IC₅₀ is strongly recommended to establish assay performance (Z-factor ≥ 0.5 required for plate acceptance); provide IC₅₀ data in same matrix if available

All samples should be shipped on dry ice in labelled, sealed cryotubes or 96/384-well plates with sealed covers. For enzymatic systems involving cofactors that degrade on thawing (e.g. SAM, NADPH), discuss cold-chain requirements with our team before shipment. Biological replicates: minimum 3 technical replicates per compound per plate recommended; minimum 2 independent biological replicates (separate enzyme preparations) for hit confirmation data intended for publication or programme advancement decisions.

Deliverables

Per-compound conversion table: substrate and product ion abundances, conversion percentage, normalised to DMSO vehicle control per plate
Ranked inhibitor list: compounds ordered by % inhibition, with accurate-mass product ion confirmation and MS2 fragmentation annotation where requested
Activity heatmap (enzyme variant or substrate scope mode): visual conversion matrix across enzyme × substrate combinations with colour-coded conversion intensity
Plate-level QC report: Z-factor per plate, coefficient of variation of control wells, signal-to-noise ratio, and pass/fail designation per plate against acceptance criteria
Dose-response curves and IC₅₀ estimates (Mode 1 follow-up): four-parameter logistic fit across 8-point dilution series for confirmed primary hits
Raw MS spectra files and extracted ion chromatograms for substrate and product ions per well
Assay condition summary: enzyme concentration, substrate concentration, incubation time and temperature, cofactor concentrations, DMSO content, MS acquisition parameters
Written interpretation report: hit identification rationale, comparison to reference inhibitor response, false-positive flag assessment, and recommended follow-up strategy

For studies requiring integration of microreactor MS screening data with cellular metabolomics or functional endpoint assays, our team can design a multi-platform campaign incorporating microfluidic chip MS formats for continuous-flow measurements alongside well-plate screening data.

Representative Results

DESI-MS enzyme screening heatmap from a 96-well plate showing substrate-to-product conversion for 80 test compounds plus controls, colour-coded from green (high conversion, no inhibition) to red (low conversion, inhibited), with hit compounds circled in blue.

96-well inhibitor screening heatmap: direct MS conversion readout

DESI-MS screening of 80 compounds against a methyltransferase enzyme in 96-well format. Each well was sampled at approximately 40 s intervals. Colour gradient: green (≥80% substrate conversion, no inhibition) to red (≤20% conversion, strong inhibition). DMSO vehicle control wells (n=8) shown in border column. Z-factor = 0.62 for this plate.

Dose-response inhibition curve from microreactor MS screening follow-up showing compound conversion percentage on y-axis versus inhibitor concentration on x-axis in log scale, with four-parameter logistic fit curve and IC50 annotation.

Dose-response confirmation: MS-derived IC₅₀ for a primary hit

Eight-concentration dose-response follow-up for a confirmed primary hit from the methyltransferase screen. Conversion percentage measured by product/substrate ratio at each concentration; IC₅₀ = 340 nM (95% CI: 210–550 nM) from four-parameter logistic fit. Product identity confirmed by MS2 fragmentation at each concentration point.

Enzyme variant activity heatmap from DiBT-MS biocatalyst screening showing a 10 x 12 grid of enzyme variants on x-axis versus substrate panel on y-axis, with colour intensity proportional to product detected by MS, identifying high-activity variants in the top-right cluster.

Enzyme variant × substrate scope heatmap: biocatalyst activity profiling

Activity heatmap from direct MS screening of 10 enzyme variants (columns) against 12 substrates (rows) in a 96-well spotted-plate DESI-MS format. Colour intensity proportional to product ion abundance detected by MS. High-activity enzyme–substrate combinations identified in cluster top-right; variant V7 shows broadest substrate scope for electron-rich substrate series.

Case Study: Direct MS Screening of Diverse Biotransformations at ~40 Seconds per Sample Enables Label-Free Enzyme Variant Discovery

Kempa E.E., Hollywood K.A., Smith C.A., Barran P.E. "Rapid Screening of Diverse Biotransformations for Enzyme Evolution." JACS Au 2021;1(5):1013–1022. https://doi.org/10.1021/jacsau.1c00027 PMC: PMC8154213.

Background

A fundamental bottleneck in directed enzyme evolution and biocatalyst discovery is the absence of label-free, high-throughput analytical methods capable of measuring diverse biotransformations across large variant panels. Fluorescence and coupled enzyme assays — the workhorses of conventional biochemical screening — are restricted to enzyme classes where the substrate or product carries a chromophore, or where a secondary reporter reaction can be coupled. This excludes a substantial fraction of synthetic biology's most valuable enzyme targets: ammonia lyases, imine reductases, kinases (measuring phosphate transfer directly), and metagenomic enzyme panels of unknown activity.

The Manchester Institute of Biotechnology team applied desorption electrospray ionisation (DESI) mass spectrometry directly to reaction spots on 96-well membranes, developing the DiBT-MS (Direct infusion of BioTransformations to Mass Spectrometry) workflow. The goal was to demonstrate that a single MS-based screening platform could assess enzyme activity across fundamentally different reaction types — kinases, imine reductases, and ammonia lyases — from crude cell lysates, without chromogenic or fluorescent substrates, and at throughputs practical for routine laboratory use.

Methods

Reactions were assembled in 96-well plates and spotted onto DESI-MS-compatible membranes. The DESI source traversed each spot at a sampling rate of approximately 40 seconds per data point. Three distinct enzyme classes were screened: (1) a panel of 11 purified sugar kinases tested against 15 monosaccharide substrates; (2) imine reductase (IRED) variants from a 384-well metagenomic library against prochiral imine substrates; and (3) phenylalanine ammonia lyase (PAL) whole-cell reaction libraries — 10 PAL variants against 15 cinnamic acid derivatives. For the PAL directed evolution application, the DiBT-MS heatmap identified high-converting variants, which were sequenced to determine amino acid changes correlated with enhanced activity toward electron-rich substrates.

Results

The DiBT-MS heatmap for the kinase panel correctly identified the known substrate specificities of all 11 enzymes within a single screening run, with product ion detection confirming phosphorylation at the expected substrate masses. For the IRED metagenomic library, the heatmap format permitted visual identification of active wells within the 384-well plate in real time, without requiring prior knowledge of which variants were active — a practical demonstration that metagenomic enzyme panels can be dereplicated by direct MS without any assay development. In the PAL directed evolution campaign, DiBT-MS identified multiple high-converting variants toward electron-rich cinnamic acid substrates including 4-fluorocinnamic acid and 4-chlorocinnamic acid — derivatives relevant to lignocellulosic biomass processing. The substrate scope revealed by MS heatmap directly guided DNA sequencing priorities, shortening the variant identification cycle.

Significance for Microreactor MS Screening

This study provides three demonstrations directly relevant to our microreactor MS screening service: first, that direct ambient MS detection of biotransformations is analytically robust across chemically diverse enzyme classes without labelling; second, that throughput of ~40 seconds per sample is sufficient for meaningful variant panels and focused inhibitor libraries in standard laboratory formats; and third, that MS heatmap output is interpretable and actionable for hit selection without statistical processing overhead. The approach exemplifies the logic of reading reaction chemistry directly — measuring product mass rather than a proxy — that underpins all modes of our microreactor MS screening service.

Figure from Kempa et al. 2021, JACS Au, showing DiBT-MS heatmap results from direct DESI-MS screening of phenylalanine ammonia lyase variants against 15 cinnamic acid substrate derivatives, with colour intensity indicating product detected by mass spectrometry across the enzyme variant panel.

Figure 3 from Kempa et al. 2021 (JACS Au, DOI: 10.1021/jacsau.1c00027, PMC8154213). DiBT-MS heatmap from PAL variant screening against 15 cinnamic acid substrates — direct DESI-MS detection of ammonia lyase products without fluorescent labelling. CC BY 4.0.

FAQ

Frequently Asked Questions

Q: What enzyme classes can be screened by microreactor MS, and are there any that are not compatible?

Any enzyme that converts a substrate to a product with a different molecular mass is compatible — which describes essentially every enzyme class. This includes methyltransferases (SAM → SAH methyl transfer), kinases (detecting phosphorylated substrate directly), ammonia lyases, halogenases, dehalogenases, acyltransferases, aminotransferases, oxidoreductases (where cofactor mass changes are detectable), and proteases (measuring cleavage product masses). The very few cases requiring special consideration are reactions where substrate and product have identical molecular formulas (e.g., pure isomerisations without any mass change) — these require a derivatisation step or an alternative detection mode. Contact our team with your enzyme's substrate–product pair for a feasibility assessment before project initiation.

Q: How does the throughput of microreactor MS compare with fluorescence-based HTS, and what scale of library is appropriate?

Ambient ionisation MS (DESI, ASAP, open-port probe) achieves throughputs of approximately one well per 10–60 seconds depending on sample complexity and instrument configuration — corresponding to roughly 1,500–8,000 data points per day. This is substantially lower than dedicated fluorescence plate readers operating at 1 second per well in full-plate mode. Microreactor MS is therefore most appropriate for focused compound libraries (hundreds to a few thousand compounds), fragment panels, compound class or analogue series assessments, and enzyme variant panels — not million-compound primary HTS campaigns. For large-scale campaigns requiring HTS-level throughput with MS detection, acoustic ejection MS or RapidFire MS platforms achieve higher rates. Microreactor MS is the strongest choice when the assay cannot be run with a spectroscopic readout, or when chemical identity confirmation of every reaction product is needed alongside activity data.

Q: Does microreactor MS screening require the enzyme to be purified?

No — this is one of the most practically important advantages of the approach. Crude cell lysates from E. coli, insect cells, mammalian cells, or yeast expressing the enzyme of interest can be used directly. The mass spectrometer identifies the product ion by its accurate mass regardless of other proteins or small molecules present in the lysate, provided the lysate does not contain a competing source of the same substrate–product conversion. We ask that customers provide basic characterisation of their lysate expression system and any known endogenous enzymatic activities that could interfere with the target reaction. Purified enzyme is preferred for dose-response inhibitor profiling (for tighter kinetic interpretation) but is not required for initial activity screening or variant panel work.

Q: How are false positives from DMSO tolerance, compound aggregation, or matrix effects controlled?

DMSO tolerance is managed by maintaining final assay DMSO concentrations at ≤1% throughout; DMSO response curves for each enzyme are established during assay development. Compound aggregation — a known source of false positives in biochemical screening — is flagged by two complementary approaches: compounds showing concentration-independent inhibition across a narrow concentration range, or inhibition patterns inconsistent with the expected IC₅₀ distribution for the enzyme class, are flagged for counter-screening in the presence of a non-ionic detergent (Tween-20 at 0.01%). Matrix-dependent signal suppression is assessed by comparing conversion values for control wells with and without the lysate matrix at identical substrate concentrations. All plate-level QC metrics (Z-factor, coefficient of variation) are reported transparently so customers can apply their own acceptance thresholds. We do not release data from plates with Z-factor < 0.5.

Q: Can microreactor MS provide kinetic data (Km, kcat) in addition to endpoint inhibition data?

Yes, with design adjustments. End-point microreactor MS screens measure conversion at a single time point — providing inhibitor rank-ordering and IC₅₀ data but not full kinetic constants. For Km and kcat determination, reactions must be sampled at multiple time points or run in a continuous-flow microreactor configuration where residence time controls the degree of conversion. Both time-course and continuous-flow kinetic modes are available within our service. If your primary goal is kinetic characterisation rather than inhibitor screening, our enzyme activity and reaction mechanism service is structured around kinetic data deliverables including Michaelis–Menten fitting, inhibition mode determination, and Lineweaver–Burk analysis.

Q: Can I send only a substrate and enzyme with no prior characterisation, and will the service still work?

Partial characterisation is acceptable — we do not require a fully optimised assay — but a minimum of two pieces of information are needed before screening begins: (1) the molecular weights of your substrate and expected product (to confirm the mass differential is sufficient for detection), and (2) evidence that the enzyme is active under at least one condition (a simple end-point assay, TLC, or chromatographic confirmation that product forms is sufficient). If neither is available and the enzyme is novel, we recommend an initial feasibility experiment (typically 2–5 days) in which we optimise buffer, pH, temperature, and cofactor conditions before committing to a full screening campaign. The feasibility experiment uses a small amount of compound (10–20 test compounds) to establish Z-factor and conversion dynamic range.

References

Kempa E.E., Hollywood K.A., Smith C.A., Barran P.E. Rapid screening of diverse biotransformations for enzyme evolution. JACS Au. 2021;1(5):1013–1022.
Knox R., Smith R., Kempa E.E., et al. Direct analysis of biotransformations with mass spectrometry — DiBT-MS. Nat Protoc. 2025;20:3295–3313.
Dueñas M.E., Peltier-Heap R.E., Leveridge M., Annan R.S., Büttner F.H., Trost M. Advances in high-throughput mass spectrometry in drug discovery. EMBO Mol Med. 2023;15(1):e14850.
Yao Z., Li Y., Xu W. Micro-immobilized enzyme reactors for mass spectrometry proteomics. The Analyst. 2025;150(14):3000–3010.

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