RedoxiFluor: A microplate assay to quantify protein thiol redox state in percentages and moles


 An accessible, time- and cost-efficient microplate assay to quantify protein thiol redox state in percentages and moles relative to the thiol proteome (i.e., context) and other targets (i.e., array mode) would be invaluable for understanding how protein thiols regulate essential biological processes. RedoxiFluor achieves several key benefits (i.e., percentages, moles, context, array mode) in a microplate format. After robustly validating RedoxiFluor, comparative analysis reveals that key benefits are intractable to other immunological techniques. Moles is an unprecedented achievement. Proof-of-concept studies illuminating fundamental redox principles (i.e., specificity, context, and heterogeneity) through measurement alone demonstrate how RedoxiFluor can advance understanding. For example, target specific protein thiol redox state changes are: (1) context specific (i.e., redox stimulus dependent); (2) selective (i.e., redox stimuli oxidise select targets); and (3) heterogenous (i.e., target responses vary markedly). RedoxiFluor is a powerful new tool for advancing a far-reaching and influential field: protein thiol redox biology.

From a measurement perspective, understanding protein thiol redox biology relies on: (1) quantifying redox state changes in percentages; (2) quantifying redox state changes in moles; (3) contextualising redox state changes relative to the thiol proteome; and (4) multiplexing proteins. Placing target thiol redox state along a set spectrum 0-100% reversibly oxidised is essential 9 . Consider two redox regulated enzymes: A and B. If A is 50% oxidised and B is 2% oxidised, a thiol reductant should only impact the activity of A. Assuming [A] and [B] are equal and similar ROS reactivity (i.e., kinetics), B should be more sensitive to a thiol oxidant since reversible oxidation occupancy can increase by 98 compared to 50%. Moles are an important redox modi able functional parameter: more of less of a [protein] can be biologically signi cant 10 . Moles rationalise percentage changes. If [A] is 50 pM and [B] is 100 pM and a redox stimulus increases the amount of reversibly oxidised protein by 5 pM, it would translate to a percent increase of 10 and 5%, respectively. Differential protein content produces a 50% difference in percentage change magnitude. Together moles and percentages can unravel threshold gating (i.e., how much of a target needs to be oxidised to exert a biologically meaningful functional effect) and rationalise the extent to which target speci c protein thiol redox state should be chemically and genetically manipulated. Context is key. Comparing target speci c protein thiol redox to the bulk thiol proteome is essential (i.e., it can reveal whether a target is highly oxidised) 11 . Finally, array mode is needed to simultaneously screen the redox state of multiple proteins to account for biological complexity 12 . For example, multiple redox switches distributed across several proteins regulate growth factor signalling [13][14][15] .
No redox proteomic or immunologically technique achieves percentages, moles, context, and array mode. Global redox proteomics achieves percentages, context, and by de nition array mode 9 . Targeted redox proteomics can achieve molar quanti cation using deuterated peptides at the expense of context and by de nition limits the number of proteins measured 16 . Coverage is often limited to abundant proteins-detecting hydrophobic and/or weakly expressed proteins is challenging 17 . Context is limited to what can be detected (i.e., global measures are derived from mass spectrometry detectable cysteines).
Even targeted redox proteomics cannot detect many potential redox regulatory thiols (e.g., 30% in the redox regulated phosphatase PTP1B 16,18 ) because they reside in di cult to digest sequences or poorly ionisable peptides 19 . Pressingly, access to redox proteomics can be rate-limiting. While readily accessible immunological techniques report the global target speci c protein thiol redox state (i.e., all modi able thiols are detected), they often cannot measure the target at all. If a target is detectable, redox analysis is usually qualitative or semi-quantitative at best 20 . When quantitative analysis is possible, it is limited to fold changes 21 . Immunological techniques lack percentages, moles, context, and array mode.
To quantify target speci c protein thiol redox state in percentages and moles relative to the thiol proteome (i.e., context) and other proteins (i.e., array mode), we present RedoxiFluor. RedoxiFluor can quickly and inexpensively deliver actionable results (i.e., a change in target speci c protein thiol redox state) in a widely accessible microplate format. The microplate format affords speci c high-throughput and automated plate reader analysis, as well as, the ability to readily calibrate the assay using known protein and redox standards. The proof-of-principles studies reported herein demonstrate the value of achieving key bene ts (i.e., percentages, moles, context, and array mode) by deriving biological insights from RedoxiFluor alone.

RedoxiFluor
RedoxiFlour uses two thiol-reactive uorescent reporters and a capture antibody functionalised solid support to measure target protein thiol redox state in percentages and moles in a microplate ( Figure 1).
Reduced thiols are labelled with a uorescent maleimide (F-MAL1) reporter via a thioether bond.
Optionally, a speci c chemotype (e.g., sulfenic acids) can be labelled using selective reductants or a direct reactivity strategy 22,23 . Direct reactivity strategies can be used to label DTT irreducible sul nic and sulfonic acids 24 . A capture antibody functionalised solid support is used to bind the target protein thiol from a biological sample (e.g., cell lysate). The thiol redox state encoded F-MAL reporters enable one to quantify protein thiol redox state in percentages (i.e., protein A mode). A biotin-conjugated detector antibody and recombinant protein standard curve (i.e., ELISA mode) enable one to calculate target speci c protein thiol redox state in percentages and moles. Global mode RedoxiFluor (i.e., untargeted F-MAL1/2 analysis) can contextualise target speci c protein thiol redox state relative to the thiol proteome. Array mode RedoxiFluor can quantify the redox state of several proteins in percentages (i.e., protein A mode). Follow-up ELISA mode RedoxiFluor can con rm and extend (i.e., percentages and moles) array mode ndings. RedoxiFluor achieves percentages, moles, context, and array mode in a widely accessible microplate format.
RedoxiFluor can quantify target speci c protein thiol redox state in percentages and moles Mixing equimolar F-MAL1/2 standards to construct arti cial redox states from 90 to 10% reversibly oxidised in L-cysteine buffer to exact any thiol dependent turn-on uorescence con rmed that RedoxiFluor can calculate redox state in percentages (supplementary Figure 1). Experiments with fully F-MAL1/2 labelled recombinant bovine serum albumin (BSA) show that RedoxiFluor can discern between different redox states from 100, 75, 50, 25, to 0% reversibly oxidised (supplementary Figure 1). No effect of uorophore labelling order is observed: the same result is obtained if F-MAL1 or F-MAL2 is used rst (supplementary Figure 1). Unbound and protein conjugated F-MAL experiments validate RedoxiFluor.
To validate RedoxiFluor using a capture antibody to bind a target from a complex biological sample, we selected the catalytic subunit (i.e., PPP2CA, uniport ID: P67775) of the serine/threonine protein phosphatase PP2A: a strategically important redox regulated target 25 . To measure PP2A, we bound a capture antibody to a protein A derivatised microplate. Protein A mode RedoxiFluor accurately (e.g., the mean observed redox difference from the standard was 0.8%) and reproducibly (e.g., the mean CV value between samples was 2.9%) quanti es the redox state of PP2A in 10-90% reversible oxidised standard samples ( Figure 2A and Table 1). Accurate and reproducible redox state reporting con rms the suitability of our labelling protocol. RedoxiFluor is speci c: no discernible F-MAL signals were observed when samples (50% F-MAL1/ 50% F-MAL2) were incubated with rabbit immunoglobin (IG) control or blank (i.e., protein A only) wells and immunodepleting PP2A abolished the signal (supplementary Figure 2). In protein A mode, RedoxiFluor consumed minimal sample (0.5-1 ug), antibody (0.1 ug per well), and rapidly delivered actionable results (i.e., 3-4 h). For context, we performed comparative macroscale and microscale immunological analysis. Click-PEG wherein clickable polyethylene glycol (PEG) payloads are used to detect reversibly oxidised thiols as mass shifted bands by immunoblot 17,26,27 cannot report PP2A redox state ( Figure 2B). PEG decorated PP2A is undetectable 20 . While ALISA works 21 , it cannot compute PP2A redox state in percentages ( Figure 2C). RedoxiFluor can quantify target protein thiol speci c redox state in percentages-a feat that proved intractable to other immunological assays.
ELISA mode RedoxiFluor can accurately (e.g., the mean observed redox difference from the standard was 1.3%) and reproducibly (e.g., the mean CV value between samples was 4.1%) ( Figure 2D and Table 1) measure the redox state of PP2A in 10-90% reversible oxidised sample standards. Speci city is evidenced by the positive biotin-conjugated detector antibody dependent signal in sample standards and lack of any discernible signal in the immunodepleted and IG controls, as well as, blanks (supplementary Figure 2). The recombinant protein standard curve evidences an ability to detect PP2A in the picogram range (i.e., from 8,000 to 125 pg/ml, supplementary Figure 2). To illustrate the signi cance, we generated new 20 and 40% reversibly oxidised redox state standards. The recombinant protein standard curve, can convert percentages into picomoles of reduced and oxidised protein. Picomoles of reduced PP2A are signi cantly greater in the 20 compared to the 40% reversibly oxidised redox state; as con rmed by a corresponding decrease in picomoles of reversibly oxidised PP2A ( Figure 2E-F). ELISA mode RedoxiFluor consumed minimal sample (0.5-1 ug), antibody (0.1 ug per well), and delivered actionable results in 12-16 h (i.e., extra time is needed to bind the capture antibody and block the plate). RedoxiFluor can quantify target speci c protein thiol redox state in percentages and moles. reported are mean (M), standard deviation (SD), the mean observed difference from the standard (i.e., 2% if a mean of 48 was registered for the 50% standard), and co-e cient of variation (CV) between standards. All values are reported in percentages. All CV values within standards (i.e., values from triplicate readings of the same sample) were all less than 5%. All standards were derived from Xenopus laevis lysates (see methods).

Mode
Protein Having established the ability of RedoxiFluor to measure the redox state of calibrated standards (i.e., knowns), we determined the redox state of experimental samples (i.e., unknowns) in a topical and translationally important immunology context. We measured the redox state of interleukin-1 receptorassociated kinase 1 (IRAK1, uniport ID: P51617) in unstimulated and lipopolysaccharide (LPS, 100 ng/ml for 30 min) stimulated human monocytes. LPS activates the innate immune response via toll-like receptors (TLR) 28 . IRAK1 helps activate TLR in response to LPS 29 . We report a potential link between the LPS induced increase in ROS and the redox state of IRAK1: a strategically important and contextually relevant protein kinase 30 . Protein A mode RedoxiFluor reveals that LPS markedly increases IRAK1 speci c reversible thiol oxidation by 33.7% from 41.05 to 74.75% ( Figure 3A-C). Note, IG controls are challenging in THP-1 cells: they express Fc binding proteins. ELISA mode RedoxiFluor con rms the dramatic (+48%) increase in IRAK1 speci c reversible thiol oxidation in LPS stimulated compared to unstimulated monocytes ( Figure 3D-F). Since both modes accurately report protein thiol redox state (Table 1), we attribute the greater increase in ELISA compared to protein A mode RedoxiFluor to baseline variability in unstimulated cells. While IRAK1 protein content remains at ~1.8 pM ( Figure 3G), reversibly oxidised IRAK1 increased by 0.9 pM in LPS stimulated compared to unstimulated controls ( Figure 3H, supplementary Figure 3). Analogous to redox proteomics and immunological techniques, the change in target speci c protein thiol redox state re ects differential oxidation formation and/or repair 3 . Despite a profound (mean change = 40.9%) increase in IRAK1 speci c reversible thiol oxidation, the thiol proteome remains highly reduced (i.e., ~85%) irrespective of LPS (supplementary Figure 4). A 15% reversibly oxidised value is consistent with previous literature 11,31 and shows that our lysis protocol limits ex vivo oxidation 32 . Global RedoxiFluor-a standalone assay-con rms the speci city of the LPS response by ruling out a global proteome wide oxidative shift. It also highlights the greater than the sum of their parts value of combining a global and speci c redox measure. RedoxiFluor discovered a new molecular feature of the innate immune response by quantifying IRAK1 redox state in percentages and picomoles.
IRAK1 RedoxiFluor realises three key bene ts (i.e., context, percentages, and moles). To achieve array mode RedoxiFlluor we con gured a microplate to simultaneously measure the redox state of several redox regulated phosphatases in unstimulated and LPS stimulated human monocytes (supplementary  Table 1). To do so, we used a cost-and time-e cient protein A microplate format. In unstimulated cells, protein phosphatase redox state ranged from 75.6 (i.e., PTP1B) to 88.2% (i.e., SHP2) reduced, with most targets clustering (i.e., within 3%) around 85%-the global protein thiol redox state. PTP1B and PP2A were, however, displaced from 85% by 10 and 6%, respectively. Array mode RedoxiFluor revealed no signi cant difference in SHP2, calcineurin, and PTEN redox state-they remained   Figure 8). RedoxiFluor can screen how multiple strategically important targets respond to a fundamental and translationally relevant redox stimulus.
Developmental biology: Macroscale RedoxiFluor reveals a fertilisation induced increase in PTEN speci c reversible thiol oxidation In the nal worked example, we studied a fundamental process: fertilisation 33 in Xenopus laevis (X. laevis). How fertilisation impacts PTEN redox state is unknown, so we measured PTEN redox state in unfertilised eggs and 1-cell zygotes (i.e., 30 min post fertilisation). Protein A mode RedoxiFluor reveals a signi cant fertilisation induced increase (+20%) in PTEN speci c reversible thiol oxidation ( Figure 5, supplementary Figure 9). In line with speci city, we observed no background F-MAL signal in IG compared to blank wells (supplementary Figure 9). When matched pair antibodies are unavailable, it can be useful to con rm microscale ndings in macroscale mode to visually verify PTEN. Macroscale RedoxiFluor is valuable because it measures a redox modi able functional property: protein interactome 3 .
To study intermolecular disul de bonds, one should covalently bind the capture antibody and complete the F-MAL labelling following elution (i.e., to preserve the bond and eliminate antibody thiols). After verifying successful PTEN "pull-down" by immunoblot (supplementary Figure 9), we measured the aggregated redox state of the PTEN interactome in a microplate (i.e., measuring the redox state of eluent aliquots). The collective redox state of the six co-eluting proteins (i.e., the PTEN interactome) is impervious to fertilisation ( Figure 5, supplementary Figure 9). However, band 2 is only present in zygotes and is largely oxidised. Consistent with the microscale protein A nding, PTEN speci c reversible thiol oxidation, as measured by analysing the eluent from the excised 55 kDa band in a plate reader, is increased (+14%) in 1-cell zygotes compared to unfertilised eggs ( Figure 5, supplementary Figure 9). Target speci c redox state can change without a corresponding redox interactome change. PTEN redox state is displaced from the thiol proteome, but this result and the accompanying redox proteomic data will be published separately in due course. One could test the redox theory of development by determining whether fertilisation induced redox changes inactivate PTEN 34 to promote PI3K signalling 33 . PTEN, an LPS unresponsive phosphatase, highlights the importance of biological context. RedoxiFluor can operate at extremes of the analytical spectrum: from the micro to macroscale.

Discussion
RedoxiFluor achieves hitherto intractable bene ts: percentages, moles, context, and array mode. Seldom have so many key bene ts been achieved in one step or in a widely accessible microplate format.
Percentages, moles, context, and array mode are inaccessible to other immunological techniques 20 . Recent technical developments enable percentage cysteine residue redox state to be quanti ed on a proteome wide scale (i.e., array and context) 9,35 . RedoxiFluor measures all target speci c redox modi able thiols; which, for many targets is a breakthrough achievement (i.e., proteomics has yet to detect 94 and 50% of the thiols in IRAK1 and PP2A, respectively). The molar bene t is unprecedented. Redox proteomic molar quanti cation is unrealised and isn't yet possible without sacri cing array and context. RedoxiFluor complements redox proteomics. Combined they can report the redox state of the target at the global (i.e., RedoxiFluor) and individual (i.e., redox proteomics) level. Combinatorial analysis could prove decisive. For example, if no redox state change occurs at mass spectrometry detectable individual thiols, RedoxiFluor can report whether undetectable residues respond. Reciprocally, if there is no global difference, redox proteomics can report whether individual thiols responded. RedoxiFluor can report redox modi able functional parameters: lifetime (i.e., moles), interactome (i.e., macroscale); locale (i.e., fractionate a sample), phase (i.e., isolate condensates), and activity (i.e., inferred from surrogate like phosphorylation). RedoxiFluor should, therefore, prove instrumental for advancing protein thiol redox biology.
The insight derived from measurement alone demonstrates how RedoxiFluor can advance protein thiol redox biology. RedoxiFluor reveals that: (1) some proteins (i.e., ITAK1) are highly oxidised (i.e., heterogeneity principle); (2) target thiol redox state is displaced from the bulk thiol proteome (i.e., speci city principle); (3) proteins, even closely related ones (i.e., SHP1 and SHP2), respond differently to the same redox stimulus (i.e., heterogeneity and speci city principles); (4) target responsiveness is stimulus dependent (i.e., context principle); (5) global thiol proteome redox state is unresponsive to a fundamental redox stimulus (i.e., speci city and preservation of redox homeostasis principles); and (6) a target speci c redox state change can occur without a change in redox state of its interactome (i.e., local speci city principle). RedoxiFluor also con rms a redox homeostasis principle: the thiol proteome is highly reduced 36 . RedoxiFluor suggests target speci c redox codes 37 : unique biological meaning might be encoded within the global response of a target protein to a redox stimulus. Consistent with different residues being oxidised between tissues and as a function of ageing 9 .
Quantitative protein thiol redox biology is insightful. The difference in the magnitude of the PP2A compared to IRAK1 LPS response (i.e., 6-8 vs 40%) might re ect a difference in protein content (i.e., ~44 vs. 2 pM). The absolute increase in reversibly oxidised PP2A is 2.5 pM greater (i.e., 3.6 vs. 0.9 pM); that there is more [PP2A] might raise (i.e., buffer) the response threshold. Moles also mean two very different percent changes (i.e., 32% difference) might both be functionally signi cant. Selecting a target to manipulate from percentages alone might be misleading. Moles are also instrumental for understanding threshold gating. If there is less of a protein in one cell compared to another, then the percent oxidised values might differ while the absolute picomoles of reversibly oxidised protein remains the same.
Practically, the percent and molar terms rationalise the extent to which target thiol redox state should be manipulated. Chemically increasing target protein thiol redox state by 40% is inappropriate if the maximal response to a redox stimulus is 20%. Finally, quantitative protein thiol redox biology is useful for mapping where target and context speci c oxidative eustress ends and distress starts 8 .
Array mode RedoxiFluor can develop data-led redox signatures of particular states (e.g., ageing) or processes (e.g., cell cycle). A redox proteomic screen to expand the survey by 1-4 orders of magnitude 9,12 can be used to rationally design and validate array mode RedoxiFluor to measure a state speci c redox signature (e.g., a redox signature of COVID-19) in a microplate. Developing multiparametric state and process speci c redox signatures could have far-reaching signi cance. For example, disease speci c redox state signatures could rapidly screen how large numbers of patients respond to a treatment; which might make it easier to evaluate the therapeutic value of next-generation antioxidants 38  Sample processing: Thiol labelling procedure Samples were incubated with 1 mM F-MAL1 or 2 (i.e., depending on the labelling order) for 30 min on ice and centrifuged at 14,000 g for 5 min at 4 o C. Soluble supernatants were passed through a 6000 kDa spin column (Bio-Rad, UK, #7326222) to remove excess F-MAL1. Flow-throughs were treated with 5 mM DTT (ThermoFisher, UK, #RO861) for 30 min on ice. After removing excess DTT with a spin column, samples were treated with 1 mM F-MAL1 or 2 for 30 min on ice. Unreacted F-MAL1 or 2 was removed with a spin column. To prepare assay calibrants, samples were lysed in 5 mM DTT for 30 min and centrifuged at 14,000 g for 5 min at 4 o C. After removing excess DTT with a spin column, samples were incubated with 1 mM F-MAL1 or 2 for 30 min. Unreacted F-MAL1 or 2 were removed with a spin column. Fully labelled F-MAL1/2 standards were mixed as appropriate to produce the 10-90% reversibly oxidised redox states (e.g., for a 10 ul nal volume, 9 ul of F-MAL1 was mixed with 1 ul of F-MAL2 to prepare the 90% reversibly oxidised standard). To prepare BSA standards, 50 ug BSA was reduced with 1 mM DTT for 15 min at room temperature (RT). Excess DTT was removed with a spin column and ow-throughs were treated with 0.5 mM F-MA1 or 2. After removing excess F-MAL1/2, the desired redox states were prepared by mixing the standards as appropriate. Samples were protected from ambient light throughout.

Isolated uorophore and recombinant BSA experiments
For the isolated uorophore experiments described in supplementary Figure 1, 1 mM of each uorophore was incubated in L-cysteine buffer (25 mM Tris, pH 7.2, 2 mM L-cysteine) for 30 min at RT in the dark to capture any thiol dependent turn-on uorescence. To construct the 90 to 10% standard curve, appropriate amounts of uorophore were mixed to a nal volume of 10 ul. Aliquots (1 ul) were analysed in triplicate in a plate reader (see RedoxiFluor analysis). BSA standards (prepared as described above) were analysed in the same way. For the gel experiments, BSA standards (1 ug) were resolved by SDS-PAGE and analysed as described below.

ELISA mode RedoxiFluor
To measure PP2A and IRAK1 redox state in ELISA mode matched pair antibodies were used. Black MaxiSorp immuno microplates (ThermoFisher, UK, #437111) were incubated with 50 ul of 2 ug/ml capture antibody overnight at 4 o C in binding buffer (35 mM NaHCO 3 , 15 mM Na 2 CO 3 , pH 9.6) on a plate shaker at 350 rpm. Unbound capture antibody was removed by washing (3 x 2 min PBST washes at 400 rpm), before wells were blocked (50% PBST, 50% Superblock) for 2 h at RT at 350 rpm and washed (3 x 2 min PBST washes at 400 rpm). The recombinant protein standards, assay calibrants (i.e., 10-90% reversibly oxidised), assay controls (i.e., immunodepleted sample, and samples (diluted as above to a nal volume of 50 ul) were added in duplicate and incubated for 2 h at RT at 350 pm in the dark. Excess sample was removed and wells were washed (3 x 2 min PBST washes at 400 rpm). Next, 0.5 ug/ml biotin-conjugated detector antibody was added for 1 h at RT at 350 rpm in the dark. After a wash step, 0.05 ug/ml of HRP-conjugated streptavidin (Abcam, UK, #ab210901) was added for 1 h at RT at 350 rpm in the dark. After a nal wash step, wells were incubated with QuantaBlu TM (ThermoFisher, UK, #15169) prepared according to the manufacturer's guidelines for 10 min at RT at 400 rpm in the dark. QuantaBlu signal was measured at 325 (excitation) and 425 (emission) nm for 100 ms on a plate reader. To stop the HRP reaction and unmask the F-MAL1/2 signal wells were incubated in denaturing buffer (4% SDS, 50 mM Tris, pH 7.1) for 15 min at RT at 500-700 rpm. After measuring F-MAL/2 uorescence in a plate reader, target speci c protein thiol redox state was calculated (see below).

Assay controls
For the PP2A and PTEN microplate experiments in X. laevis described, sample aliquots (diluted as above) were also added to rabbit isotype control wells in protein A and ELISA mode to assess non-speci c binding. To check speci city in the case of PP2A, samples were incubated with a PP2A capture antibody functionalised protein A magnetic beads (see below) overnight at 4 o C with gentle rotation. The PP2A immunodepleted sample was added to a second capture antibody functionalised magnetic bead for 1 at RT before being added to the protein A or ELISA microplate. Sample speci c (i.e., THP-1 for IRAK1) calibrated standards (e.g., 10-90% reversibly oxidised) were used in every RedoxiFluor experiment.

Array mode RedoxiFluor
To assess the redox state of multiple targets in array mode, 0.1 ug of SHP1 (row B), SHP2 (row C), PTP1B (row D), PP2A (row E), PTEN (row F), CD45 (row G), and calcineurin (row G) capture antibodies (see Table 3) were added in binding buffer (50%: PBST; 50% Superblock) to a black protein A derivatised microplate for 1 h at RT at 350 rpm on a plate shaker. Row A was reserved as a blank well. Unstimulated (lanes 1-3 & 7-9) and LPS stimulated (lanes 4-6 & 10-12) samples (diluted 1:10) were added for 2 h at 350 rpm in the dark. Thereafter array mode experiments were identical to protein A mode RedoxiFluor.

Global thiol proteome redox state
To measure the redox state of the thiol proteome, samples (1 ul diluted in 199 ul PBS) were measured in triplicate in a plate reader.

Macroscale: RedoxiFluor
Aliquots (10 ug) of PTEN capture antibody or rabbit isotype control (ThermoFisher, UK, #31235) were incubated with 50 ul protein A derivatised magnetic beads for 1 h at RT. Beads were magnetised to remove excess capture antibody and incubated with undiluted X. laevis samples (~500 ug protein) overnight at 4 o C. Beads were magnetised to remove unbound sample, washed in PBST (0.025% Tween- 20), and resuspended in denaturing buffer (4% SDS, 200 mM Tris, 20% glycerol, pH 7.1). To prevent any potential heating induced uorescence artefacts, PTEN was eluted from the capture antibody by chemically breaking the antibody apart: the disul de bonds required to structure the antibody for target binding were reduced with 10 mM DTT for 15 min at RT. This procedure proved effective as magnetic beads were negative for F-MAL1-2 following elution. Note, glycine elution is incompatible with RedoxiFluor (the low pH destroys the uorescence). Eluents were isolated by magnetising the beads. To assess PTEN interactome redox state, triplicate eluent aliquots (2 ul) were measured in a plate reader. To identify the interacting proteins, eluents were resolved by SDS-PAGE using a pre-cast 4-15% gradient gel (Bio-Rad, UK, #4561085) and F-MAL1/2 signals were captured on a gel scanner for 1 s using the appropriate lters. To assess PTEN redox state, eluents were resolved by SDS-PAGE and the speci c PTEN band (as con rmed by immunoblot, see below) was manually excised and passively eluted in distilled water (dH 2 O) for 24 h at RT with vigorous agitation. Passive elution was successful because the bands were F-MAL1/2 negative after 24 h. For PTEN speci c redox analysis, no gel scanner imaging was performed to eliminate potential photo-toxicity interfering with microplate analysis. Passively eluted PTEN redox state was measured using a plate reader.

RedoxiFluor analysis
Irrespective of the RedoxiFluor mode (i.e., protein A mode), target redox state was assessed by measuring F-MAL1 (494-518 nm) and F-MAL2 (651-671 nm) with a 5 nm bandwidth for 100 ms on a plate reader. After blank subtraction, F-MAL1 and F-MAL2 signals (v) were corrected using the following equation: v = (v/E)/q or a derivative thereof were E and q represent the extinction co-e cient and quantum yield, respectively. Corrected values were totalled and precent reduced or reversibly oxidised protein was calculated (i.e., reduced = (reduced/total)*100). In ELISA mode RedoxiFluor, pg/ml protein content values computed from the recombinant protein standard curve were converted to picomoles by dividing them by the molecular weight of the target protein (i.e., picomoles = picogram / target molecular weight). The amount of reduced and reversibly oxidised target protein in picomoles could then be calculated (i.e., pM reduced = total pM*reduced%).

Immunoblot
After resolving samples by SDS-PAGE, they were transferred to a 0.45 uM 100% methanol activated PVDF membrane for 1 h. Membranes were blocked for 1 h in 5% non-fat-dry milk (NFDM) and incubated with a

Statistics
Data-set normality was assessed using Shapiro-Wilk and Kolmogorov-Smirnov testing. For within sample redox state (i.e., reduced vs. reversibly oxidised), data were analysed using a paired t-test or nonparametric equivalent. For between samples redox state (i.e., %reversibly oxidised), data were analysed with an unpaired t-test or non-parametric equivalent. When comparing multiple proteins in the array to determine how their reversible oxidation set-points differed, a one-way ANOVA was used. In all cases, alpha was set to P > 0.05 and tests were performed on GraphPad Prism Version 9 (https://www.graphpad.com).  General microscale RedoxiFluor scheme. Block and wash steps are omitted for clarity throughout (see methods). Step 1. Bind the capture antibody to a microplate. Step 2. Bind the uorescent maleimide (F-MAL) decorated target. In two colour mode, one spectrally distinct reporter labels reduced thiols (e.g., F-MAL1, green groups) and another reversibly oxidised thiols (e.g., F-MAL2, red groups). Step 3. The target is eluted from the capture antibody to unmask the F-MAL reporters.
Step 4. Target speci c F-MAL1/2 signals are measured in a plate reader.

Figure 5
Developmental biology: Micro and macroscale protein A mode RedoxiFluor reveals a fertilisation induced increase in PTEN speci c reversible thiol oxidation. A. Protein A mode RedoxiFlour reveals a signi cant (unpaired t-test, P = 0.0002 n = 6) increase in PTEN speci c reversible thiol oxidation in 1-cell zygotes compared to unfertilised eggs in X. laevis. B. Macroscale RedoxiFluor nds no signi cant unpaired t-test, P = 0.2592, n = 3) difference in PTEN interactome redox state in 1-cell zygotes compared to unfertilised eggs in in X. laevis. C. A representative SDS-PAGE gel image showing the PTEN (highlighted) and the redox state (reduced = green channel; oxidised = red channel) of its interactome (arrows 1-6 corresponding to proteins with a molecular weight of ~100, 75, 60, 37, 25 and 10 kDa respectively) in unfertilised eggs (E) and zygotes (Z). D. Quantifying the redox state of the PTEN speci c band manually excised and eluted from (C) in unfertilised eggs and 1-cell zygotes in X. laevis con rms the signi cant (unpaired t-test, P = 0.0020 n = 3) fertilisation induced increase in PTEN speci c reversible thiol oxidation.

Supplementary Files
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