A method of detecting protein tyrosine phosphatases in a sample

Abstract

The present invention relates to the field of proteomics and analytical biochemistry, specifically to methods for detecting and quantifying protein tyrosine phosphatases (PTPs) in samples. More particularly, the present invention relates to the use of high-throughput liquid chromatography–tandem mass spectrometry (LC-MS / MS) methods employing scheduled Parallel Reaction Monitoring (sPRM) for determining both expression levels and enzymatic activity of individual PTP isoforms, and the use of such methods in the diagnosis, prognosis, and monitoring of disease and conditions associated with aberrant PTP expression and / or activity levels.

Description

[0001] A METHOD OF DETECTING PROTEIN TYROSINE PHOSPHATASES IN A SAMPLE

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003]

[0001] This application claims the benefit of priority of Greece Patent Application No. 20240100571 filed 13 August 2024, the content of which being hereby incorporated by reference in its entirety for all purposes.

[0004] TECHNICAL FIELD

[0005]

[0002] The present invention relates to the field of proteomics and analytical biochemistry, specifically to methods for detecting and quantifying protein tyrosine phosphatases (PTPs) in samples More particularly, the present invention relates to the use of high-throughput liquid chromatography-tandem mass spectrometry (LC-MS / MS) methods employing scheduled Parallel Reaction Monitoring (sPRM) for determining both expression levels and enzymatic activity of individual PTP isoforms, and the use of such methods in the diagnosis, prognosis, and monitoring of disease and conditions associated with aberrant PTP expression and / or activity levels

[0006] BACKGROUND

[0007]

[0003] Protein Tyrosine Phosphatases (PTPs) are a family of enzymes that play critical roles in cellular signal transduction by catalysing the dephosphorylation of phosphorylated tyrosine residues on proteins. In the human genome, thirty-seven distinct PTPs have been identified. These enzymes work in concert with tyrosine kinases to regulate the dynamic phosphorylation-dephosphorylation cycles that underpin essential biological processes including cell growth, differentiation, and immune responses.

[0008]

[0004] The proper functioning of PTPs ensures the timely attenuation of tyrosine phosphorylation signals, thereby resetting signalling pathways and allowing repeated cycles of activation. Dysregulation of this control mechanism has been implicated in a range of pathologies, notably various metabolic diseases and cancers, where aberrant phosphotyrosine signalling is a hallmark.

[0009]

[0005] While gene expression levels provide some indication of potential PTP activity, they do not reliably reflect functional enzyme activity. This is due, in part, to the fact that PTP activity is not solely governed by protein abundance but also by post-translational modifications such as oxidation. Specifically, oxidation of a critical cysteine residue within the catalytic site can render the enzyme inactive, regardless of expression level. As such, functional readouts of PTP activity are essential for understanding the biological role and therapeutic relevance of individual PTPs.

[0010]

[0006] Despite their importance, current technologies offer limited capabilities in accurately assessing the activity of specific PTPs. Genomic analyses provide no functional information, and conventional proteomics approaches typically lack the sensitivity, specificity, or throughput to discriminate among the 37 individual PTP isoforms. Moreover, there are currently no scalable, high- throughput methods that allow for the exclusive and parallel detection, identification, or quantification of individual PTPs in complex biological samples. This hinders both basic research and clinical translation, as it remains unclear which PTPs are functionally relevant in a given pathological context and should be targeted for therapeutic intervention.

[0011]

[0007] Therefore, there is still need in the art for a method to address the drawbacks of existing approaches. In particular, there is a need in the art for methods that may enable the detection and, optional quantification, of the expression level and / or enzymatic activity of protein tyrosine phosphatases (PTPs) in a sample.

[0012] SUMMARY

[0013]

[0008] In one aspect there is provided a method of detecting, and optionally quantifying, the expression level and / or enzymatic activity of phosphatases (PTPs) in a sample containing, or suspected of containing, PTPs, comprising the following steps: a) processing the sample; b) isolating PTPs in the sample; and detecting, and optionally quantifying, the expression level and / or enzymatic activity of the PTPs in the sample using LC-MS / MS analysis in scheduled PRM mode.

[0014]

[0009] In various embodiments, the expression level and / or enzymatic activity of the PTPs identified, and optionally quantified, in the sample, is compared to the expression level and / or enzymatic activity of the PTPs identified, and optionally quantified, in a control or comparative sample.

[0015]

[0010] In various embodiments, step a) comprises converting PTPs in the sample into an inactive sulfonic acid state (S-O3H).

[0016]

[0011] In various embodiments, step a) comprises chemical derivatization of the sample with a reducing agent and / or an alkylating agent.

[0017]

[0012] In various embodiments, the chemical derivatization converts both total PTPs and oxidized PTPs within the sample into S- state, with subsequent hyperoxidation carried out to convert the S- state PTPs into an inactive sulfonic acid state (S-O3H).

[0018]

[0013] In various embodiments, step a) comprises dividing the sample into two pools of sample pool (A) for obtaining total PTPs and sample pool (B) for obtaining oxidized PTPs.

[0019]

[0014] In various embodiments, sample pool (A) and (B) are treated with lysis buffers, preferably the lysis buffer used for sample pool (A) is distinct from the lysis buffer used for sample pool (B), more preferably the lysis buffer used for sample pool (A) comprises dithiothreitol (DTT) and the lysis buffer used for sample pool (B) comprises N-Ethylmaleimide (NEM).

[0020]

[0015] In various embodiments, following treatment with the lysis buffers, sample pool (A) and (B) are hyperoxidated, preferably pervanadate is mixed with each sample pool (A) and (B) under suitable conditions to convert all PTPs into a permanently inactive sulfonic acid state (S-O3H).

[0021]

[0016] In various embodiments, the isolation of the PTPs comprises antibody-based immunoprecipitation, preferably the antibody is specific for PTPs in an oxidized state, preferably the antibody is an ox-PTP antibody and the PTPs are in an inactive sulfonic acid state (S-O3H), and the ox-PTP antibody is specific for sulfonic acid (S-O3H), more preferably the immunoprecipitation comprises crosslinking of the ox-PTP antibody.

[0022]

[0017] In various embodiments, step a) further comprises a step of protein digestion and peptide purification of the sample.

[0023]

[0018] In various embodiments, step c) comprises detecting, and optionally quantifying, the enzymatic activity of PTPs in the sample, and subtracting the oxidized PTP from total PTP, wherein the oxidized PTPs indicate the number of inactive PTPs in the sample with the remaining PTPs in the total PTPs indicating the number of active PTPs in the sample.

[0024]

[0019] In various embodiments, the LC-MS / MS analysis comprises a reverse-phased chromatography, preferably using a UHPLC system, more preferably the LC comprises a 75 minute gradient.

[0025]

[0020] In various embodiments, step c) comprises detecting, and optionally quantifying, one or more PTP peptides in the sample, preferably the one or more PTP peptides comprise a PTP catalytic site, wherein the PTP catalytic site comprises a hyperoxidized cysteine residue in the PTP catalytic site.

[0026]

[0021] In various embodiments, the scheduled PRM mode uses pre-defined and selected mass-to- charge ratios (m / z) and retention time (RT) windows for the PTPs, preferably for PTP peptides.

[0027]

[0022] In various embodiments, the retention time is configured to prevent noise peaks from near - isobaric peptide ions, and to increase the number of transitions that can be obtained for each peptide, enhancing quantification.

[0028]

[0023] In various embodiments, mass-to-charge ratios for +2, +3, and +4 charge states of the PTP peptides are used for the scheduled PRM.

[0029]

[0024] In various embodiments, step c) further comprises distinguishing between each PTP identified in the sample.

[0030]

[0025] In various embodiments, a precursor ion spectrum and / or a fragment ion spectrum is obtained from the LC-MS / MS run to detect, and optionally quantify, the expression level and / or enzymatic activity of PTPs in the sample.

[0031]

[0026] In various embodiments, the PTPs are human PTPs, and the method is capable of detecting and distinguishing between all 37 human PTPs.

[0032]

[0027] In various embodiments, parameters of the LC-MS / MS analysis in scheduled PRM mode are configured to detect and distinguish between human PTPs based on a PTP amino acid sequence upstream of a PTP catalytic site comprised in PTP peptides, preferably the parameters include: (I) selected PTP peptide sequences corresponding to each human PTP, preferably the PTP peptide comprise the PTP amino acid sequence upstream of the PTP catalytic site;(ii) modification of the PTP peptide sequence (i), preferably modify a cysteine (C) residue in the PTP catalytic site to add 48 Daltons (Da) to the molecular weight of the peptide (i)); (iii) charge state (z) for each PTP peptide sequence (i); (iv) precursor m / z of each PTP peptide sequence (i); (v) retention time of each PTP peptide sequence (i); (vi) adjusted retention time; and / or (vii) minimum start time and minimum end time related to the RT.

[0033]

[0028] In various embodiments, the parameters of the LC-MS / MS analysis in scheduled PRM mode of the human PTPs, are defined in Table 5, such that the method is capable of detecting and distinguishing between 37 human PTPs in a sample.

[0034]

[0029] In various embodiments, the sample is a biological sample that has been obtained from a subject, and the expression level and / or enzymatic activity of PTPs identified, and optionally quantified, in the biological sample is compared with reference expression levels and / or enzymatic activity of PTPs associated with a disease or condition associated with one or more PTPs.

[0035]

[0030] In various embodiments, each reference expression levels and / or enzymatic activity is specific for a disease or condition associated with one or more PTPs.

[0036]

[0031] In a second aspect there is provided a method for the diagnosis, prognosis, or monitoring of a disease or condition associated with one or more PTPs in a subject, comprising detecting, and optionally quantifying, the expression level and / or enzymatic activity of protein tyrosine phosphatases (PTPs) in a biological sample that has been obtained from the subject, using the method disclosed herein, wherein the detected, and optionally quantified, expression level and / or enzymatic activity of protein tyrosine phosphatases (PTPs) assists in the diagnosis, prognosis, or monitoring of a disease or condition associated with one or more PTPs.

[0037]

[0032] In a third aspect there is provided a method for assisting in determining a subject's risk for developing a disease or condition associated with one or more PTPs, comprising detecting, and optionally quantifying, the expression level and / or enzymatic activity of protein tyrosine phosphatases (PTPs) in a biological sample that has been obtained from the subject, using the method disclosed herein, wherein the detected, and optionally quantified, expression level and / or enzymatic activity of protein tyrosine phosphatases (PTPs) is indicative of the subject's risk for developing a disease or condition associated with one or more PTPs.

[0038]

[0033] In a fourth aspect there is provided a method for predicting a subject' s response to a therapeutic treatment for treating a disease or condition associated with one or more PTPs, comprising detecting, and optionally quantifying, the expression level and / or enzymatic activity of protein tyrosine phosphatases (PTPs) in a biological sample that has been obtained from the subject, using the method disclosed herein, wherein the detected, and optionally quantified, expression level and / or enzymatic activity of protein tyrosine phosphatases (PTPs) is predictive of the subject's response to the therapeutic treatment.

[0039]

[0034] In a fifth aspect there is provided a method for monitoring treatment efficacy in a subject undergoing treatment for a disease or condition associated with one or more PTPs comprising, detecting, and optionally quantifying, the expression level and / or enzymatic activity of protein tyrosine phosphatases (PTPs) in a biological sample that has been obtained from the subject at different time points before, during, or after treatment, using the method disclosed herein, optionally, the detecting step is repeated two or more times before, during, or after treatment within a time-frame and the detection at each time point is compared against each other to assess the progression of the disease or condition and efficacy of the treatment.

[0040]

[0035] In various embodiments, the disease or condition associated with one or more PTPs are known to be associated with aberrant expression and / or activity level of one or more PTPs, and comprises cancer, a metabolic disorder, an autoimmune disease, a neurological disorder, an infectious disease, a cardiovascular disease, a developmental disorder, and an inflammatory disease.

[0041]

[0036] In a sixth aspect there is provided a method for developing or identifying therapeutic agents capable of modifying the expression and / or activity level of PTPs in a subject, the method comprising: a) measuring the expression level and / or enzymatic activity of protein tyrosine phosphatases (PTPs) in a first biological sample obtained from the subject, with the method disclosed herein; b) administering a candidate therapeutic agent to the subject; c) measuring the expression level and / or enzymatic activity of protein tyrosine phosphatases (PTPs) in a second biological sample obtained from the subject after step b), with the method disclosed herein, following the administration of the candidate agent; d) characterizing the candidate therapeutic agent as capable of modifying the activity of PTPs, if the subject's measured expression level and / or enzymatic activity of PTPs increases or decreases following administration of the candidate therapeutic agent.

[0042] BRIEF DESCRIPTION OF THE DRAWINGS

[0043]

[0037] Various embodiments will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings.

[0038] FIG. 1 shows a graphical summary of the series of steps involved in detecting and screening for protein expression and enzymatic activity of all 37 human protein tyrosine phosphatases (PTPs). The table illustrated in the LC-MS / MS step is the same as Table 5 disclosed herein.

[0044]

[0039] FIG. 2 shows a schematic workflow for the chemical derivatization step shown in FIG.1 .

[0045]

[0040] FIG. 3 shows the number of detected synthetic PTP peptides with probabilities of hyperoxidation.

[0046]

[0041] FIG. 4A-C shows PTPRH synthetic peptide detection: FIG. 4A Chromatographic peaks with different shades indicating the detected y and b fragment ions; FIG. 4B Dot product (dotp) values with different shades indicating the detected y and b fragment ions; and FIG. 4C Mirror spectra with peptide coverage with DDA and scheduled-PRM acquisitions for PTPRH synthetic peptide (SEQ ID NO: 45). The black lines represent y and b ions matched to the peptide sequence shown on the right.

[0047]

[0042] FIG. 5A-C shows PTPRC synthetic peptide detection: FIG. 5A Chromatographic peaks with different shades indicating the detected y and b fragment ions; FIG. 5B Dot product (dotp) values with different shades indicating the detected y and b fragment ions; and FIG. 5C Mirror spectra with peptide coverage with DDA and scheduled-PRM acquisitions for PTPRC synthetic peptide (SEQ ID NO: 3). The black lines represent y and b ions matched to the peptide sequence shown on the right.

[0048]

[0043] FIG. 6 shows Western blot results for hyperoxidation in cancer cell lines and liver organoid (TOT: total PTP; OX: inactive PTP).

[0049]

[0044] FIG. 7A-I shows a data quality assessment for PTPN1 (SEQ ID NO:1 ) and PTPRA (SEQ ID NO: 10), where different shading indicate the detected y and b fragment ions: FIG. 7A,D,G Chromatographic peaks showing retention time and peptide-spectrum matches (PSMs / IDs) for PTPN1 and PTPRA; FIG. 7B Mirror plots comparing ion spectra from biological samples (top) and the synthetic PTPN1 peptide (bottom), alongside the sequence ion coverage. The black lines represent y and b ions matched to the peptide sequence shown on the right; FIG. 7C,E,H Peak area percentages for all three replicates in each sample, along with dotp values indicating similarity in product ion ratio; and FIG. 7F,I Mirror plots comparing ion spectra from normal (top) and the tumor liver organoids (bottom), alongside the sequence ion coverage for cell lines. The black lines represent y and b ions matched to the peptide sequence shown on the right.

[0050]

[0045] FIG. 8A-C shows PTP detection in biological samples in scheduled-PRM: FIG. 8A Number of detected PTP proteins in DDA (Hela only) and scheduled-PRM modes. One sequence was considered for the count of PTP proteins. Light colour: inactive PTPs; Dark colour: total PTPs; FIG. 8B Distribution of detected PTPs across cell lines (Active: Top; Inactive: down); and FIG. 8C Phosphatases detected in liver organoid. Normal: Adjacent non-tumor tissue, Tumor: Cancerous tissue.

[0051] DETAILED DESCRIPTION

[0052]

[0046] The following detailed description refers to, by way of illustration, specific details and embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. Other embodiments may be utilized and structural and logical changes may be made without departing from the scope of the invention. Embodiments described below in context of the methods are analogously valid for the respective kits, and vice versa. The various embodiments are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments to form new embodiments.

[0053]

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "comprises" means "includes." In case of conflict, the present specification, including explanations of terms, will prevail. “About”, as used herein in connection with numerical values refers to the referenced numerical value ±10% or ±5%.

[0054]

[0048] The present inventors discovered that scheduled Parallel Reaction Monitoring (PRM) mode may be advantageously applied to liquid chromatography-tandem mass spectrometry (LC-MS / MS ) analysis of a sample for the purpose of detecting, identifying, and distinguishing, protein tyrosine phosphatases (PTPs) in the sample. Moreover, the application of scheduled PRM mode may be used to quantify the expression level and / or enzymatic activity of PTPs in a sample. Surprisingly, it was discovered that the application of scheduled PRM over traditional Data- Dependent Acquisition (DDA) or PRM, may not only enable the identification of all PTPs in a sample but also minimizes noise peaks from interferences and increase in the number of transitions for each peptide. This results in higher intensities and enhanced sensitivity, ensuring precise and optimal quantification.

[0055]

[0049] Accordingly, there is provided a method of detecting, and optionally quantifying, the expression level and / or enzymatic activity of protein tyrosine phosphatases (PTPs) in a sample containing, or suspected of containing, PTPs, that may comprise the steps of: processing the sample and isolating PTPs in the sample; and detecting, and optionally quantifying, the expression level and / or enzymatic activity of the PTPs in the sample using LC-MS / MS operated in scheduled PRM mode.

[0056]

[0050] As used herein, the term “protein tyrosine phosphatases (PTPs)” refers to a large family of transmembrane and intracellular enzymes that dephosphorylate substrates involved in a variety of regulatory processes (Fischer et al., 1991 , Science 253:401 -406). PTPs play an important role in the regulation of phosphorylation of proteins and represent the counterparts of kinases. The PTPs superfamily includes cysteine-, aspartic acid- or histidine- based phosphatases. Cysteine-based phosphatases represent up to 85% of PTP superfamily and can be further subdivided into different categories. These categories include classical PTPs, dual-specificity phosphatases (DSP), SAC phosphoinositide phosphatases, PTP-like phytases, (PALD1 ), INPP4 phosphatases, TMEM55 phosphatases, low molecular weight PTPs (LMW), SSU72 phosphatases and CDC25 phosphatases. Among classical PTPs, there are two types : (i) non-receptor or intracellular PTPs and (ii) receptorlike or transmembrane PTPs. Intracellular PTPs contain one catalytic domain only, whereas most receptor-like enzymes contain two. The catalytic domain consists of about 250 amino acids (Niels Peter Hundahl Moller et al. Protein tyrosine phosphatases (PTPs) as drug targets: Inhibitors of PTP- 1 B for the treatment of diabetes; Current Opinion in Drug Discovery & Development 3(5), 527-540 (2000)). The classical PTPs, exclusively dephosphorylate tyrosine residues. The PTPs may include endogenously oxidised PTPs.

[0057]

[0051] In various embodiments, the PTPs are human PTPs. in various embodiments, the human PTPs comprise at least one human PTP selected from the PTPs listed and detailed in below Table 1, or ail human PTPs listed and detailed in below Table 1.

[0058]

[0052] Table 1 : Lisi of Human PTPs and reference information

[0059]

[0053] in various embodiments, the method disclosed herein may detect, identify and distinguish at least one or a set of PTPs in a sample, in various embodiments, the method may detect, identify and distinguish gi; 37 human PTPs in a sample. Specifically, the method may detect, identify and distinguish the group of PTPs comprising PTPRA, PTPRB. PTPRC, PTPRD, PTPRE, PTPRF, PTPRG, PTPRH, PTPRJ, PTPRK, PTPRM, PTPRN, PTPRN2, PTPRO, PTPRQ, PTPRR, PTPRS, PTPRT, PTPRtJ, PTPRZ. PTPN1 , PTPN2. PTPN3, PTPN4, PTPN5. PTPN6, PTPN7, PTPM9, PTPN11 , PTPN12, PTPN13, PTPN14, PTPN18, PTPN20, PTPN21 , PTPN22 and PTPN23 in a sampie

[0060]

[0054] in various embodiments, the method may detect, identify P t PN1 , optionally in a set of one or more other PTPs.

[0055] As used herein, the term “sample” refers to any sample for which the identification of protein tyrosine phosphatases (PTP), is desired to be obtained. Thus, the sample may be any sample that contains biological material and which contains or is suspected to contain an active PTP. The sample may include but is not limited to: an established cell line; a cell culture, including a primary cell culture; a biological fluid such as plasma or blood; a tissue sample; or a tissue extract; or an organoid. The sample may be human or non-human in origin, or may contain human or non-human PTPs. The sample may be any sample that can be obtained by invasive or non-invasive techniques from a human subject. Such samples may be obtained by any standard method known in the art, e.g., a biopsy from a tumour. The sample may be a normal sample (for example, healthy or non-diseased) or a diseased sample (for example a sample taken from a tumour or from a subject suffering from a disease such as cancer, a metabolic disease or condition, a brain disease including Alzheimer's disease, a viral infection, or any other disease, or a subject suspected of suffering from such a disease). The sample may be from a biopsy of a tumour, including a tumour that may be suspected of having metastasised from a different location than the biopsy site. The sample may be a sample that has been exposed to a drug treatment for disease, including a combination drug treatment, including exposed to one or more PTP inhibitors, or may be free from exposure to such treatment.

[0061]

[0056] In various embodiments, the sample may be any biological sample from a subject in which the individual expression levels and / or enzymatic activity of the PTPs can be determined. In various embodiments, the biological sample is a tissue sample, a stool sample, a cell sample or a bodily fluid sample. In a further embodiment, the biological sample is a tissue sample, in particular a neoplastic tissue sample, such as a tumour sample. The biological sample may also be derived from a biological fluid or body fluid, for example, whole blood, blood, urine, lymph fluid, serum, plasma, nipple aspirate, ductal fluid, saliva, bile, sputum or tumour exudate.

[0062]

[0057] In various embodiments, the sample is a tissue sample derived from fine-needle aspirate. In various embodiments, the tissue sample is a resected tissue sample. In various embodiments, the sample is a tissue biopsy or tissue fine-needle aspirate, for example a tumour tissue biopsy or tumour fine-needle aspirate from a primary tumour tissue or a metastatic tumour tissue. In various embodiments, the sample is resected tumour tissue, e.g. resected primary or metastatic tumour tissue. The biological sample can be obtained from the subject in any way typically used in clinical settings for obtaining a sample comprising the required cells or proteins. For example, the sample can be obtained from fresh, frozen, or paraffin-embedded surgical sample or biopsies of an organ or tissue comprising the suitable cells or proteins to be tested, for example a formalin-fixed, paraffin- embedded (FFPE) sample. If desired, the sample can be mixed with a fluid or purified or amplified or otherwise treated. For example, the sample may be treated in one or more purification steps in order to increase the purity of the desired cells or proteins in the sample, or they may be examined without any purification steps. In various embodiments, the sample may be a fresh sample or a frozen sample. In various embodiments, the sample may comprise cells, organoids, or tissues. In various embodiments, the sample may be snap-frozen immediately after collection and stored at -80°C until further processing to prevent any potential protein degradation.

[0063]

[0058] In various embodiments, the step of processing the sample may comprise performing chemical derivatization of the sample in order to convert PTPs present in the sample into an inactive sulfonic acid state (S-O3H). The chemical derivatization to sulfonic acid (S-O3H) chemically locks the PTPs in the sample into a stable, inactive state with a unique mass signature, enabling highly specific identification and quantification of individual PTPs by mass spectrometry.

[0064]

[0059] In various embodiments, the chemical derivatization converts both total PTPs (i.e. S- state and S-OH state) and oxidized PTPs (i.e. S-OH state) within the sample into S- state. In various embodiments, the sample may then be hyperoxidated to convert the S- state of both the total PTPs and oxidized PTPs to reach the inactive sulfonic acid state (S-O3H).

[0065]

[0060] In various embodiments, the chemical derivatization comprises mixing and treating the sample with a reducing agent, and / or an alkylating agent.

[0066]

[0061] In various embodiments, the alkylating agent may comprise N-ethylmaleimide (NEM), iodoacetamide (IAM), iodoacetic acid (IAA), acrylamide, methyl methanethiosulfonate (MMTS), vinylpyridine, chloroacetamide (CAM), a maleimide derivative or combinations thereof. In various embodiments, the alkylating agent may be N-ethylmaleimide (NEM).

[0067]

[0062] In various embodiments, the reducing agent may comprise dithiothreitol (DTT), tris(2- carboxyethyl)phosphine (TCEP), p-mercaptoethanol, dithiobutylamine, cysteine, glutathione, or sodium borohydride or combinations thereof. In various embodiments, the reducing agent may be dithiothreitol (DTT).

[0068]

[0063] In various embodiments, the reducing agent, and / or alkylating agent may be comprised in a lysis buffer to mix and contact the sample.

[0069]

[0064] As used herein, the term “lysis buffer" refers to a chemical solution used to disrupt / lyse cells and solubilize proteins while preserving the biochemical state of protein tyrosine phosphatases (PTPs) during sample processing and for chemical derivatization and subsequent mass spectrometric analysis. In addition to the reducing agent, and / or alkylating agent, the lysis buffer may further comprise one or more of the following components: (i) a chaotropic agent selected from urea, thiourea, or guanidine hydrochloride; (ii) a buffering agent such as ammonium bicarbonate, Tris-HCI, HEPES, or phosphate buffers: (iii) a detergent selected from sodium deoxycholate, sodium dodecyl sulfate (SDS), Triton X-100, NP-40, or CHAPS, preferably sodium deoxycholate; (iv) one or more protease inhibitors including but not limited to AEBSF, aprotinin, leupeptin, pepstatin, or E64; and (v) nucleases such as DNase I or RNase A. The lysis buffer may be suitably formulated to promote efficient protein extraction, stabilize PTP oxidation states, prevent enzymatic degradation, and ensure compatibility with downstream chemical derivatization and LC-MS / MS analysis.

[0070]

[0065] In various embodiments, the lysis buffer comprises (i) a reducing agent and / or alkylating agent, (ii) a chaotropic agent, preferably urea, (iii) a buffering agent, preferably AmbiC, (iv) protease inhibitors, (v) one or more nucleases, preferably DNAse and RNAse, and (vi) a buffering agent, preferably sodium deoxycholate.

[0071]

[0066] In various embodiments, the lysis buffer comprises a reducing agent. In various embodiments, the lysis buffer comprises (I) a reducing agent, (ii) a chaotropic agent, preferably urea, (iii) a buffering agent, preferably AmbiC, (iv) protease inhibitors, (v) one or more nucleases, preferably DNAse and RNAse, and (vi) a buffering agent, preferably sodium deoxycholate. This lysis buffer comprising the reducing agent may be termed herein as lysis buffer (A), or a first lysis buffer.

[0072]

[0067] In various embodiments, the lysis buffer comprises an alkylating agent. In various embodiments, the lysis buffer comprises (I) an alkylating agent, (ii) a chaotropic agent, preferably urea, (iii) a buffering agent, preferably AmbiC, (iv) protease inhibitors, (v) one or more nucleases, preferably DNAse and RNAse, and (vi) a buffering agent, preferably sodium deoxycholate. This lysis buffer comprising the alkylating agent may be termed herein as lysis buffer (B), or a second lysis buffer. In various embodiments, after mixing of the lysis buffer with the sample, a reducing agent may be added to the mixture.

[0073]

[0068] In various embodiments, after mixing and treating with the lysis buffer, the method may comprise a buffer exchange step to remove interfering small molecules. The buffer exchange step may remove one or more of the following components: urea, chaotropic agents, detergents, reducing agents, alkylating agents, salts, and other low molecular weight contaminants that may interfere with protease activity or mass spectrometric detection. Suitable buffer exchange methods include, but are not limited to, size exclusion chromatography, spin column filtration, dialysis, ultrafiltration, or solidphase extraction. The buffer exchange may ensure optimal conditions for proteolytic digestion, maintains the stability of the chemical derivatization products, and enhances the sensitivity, accuracy, and reproducibility of subsequent LC-MS / MS analysis.

[0074]

[0069] In various embodiments, the step of processing the sample comprises dividing the sample into two pools of sample pool (A) for obtaining total PTPs and sample pool (B) for obtaining oxidized PTPs. Chemical derivatization may be carried out on each sample pool (A) and (B) separately to convert the total PTPs and oxidized PTPs present into an inactive sulfonic acid state (S-OsH). In various embodiments, the sample may be equally divided into sample pool (A) and (B). In this context, “equally divided” refers to dividing the sample into two portions of substantially equal volume or protein content to ensure equivalent representation of all PTPs in each pool for accurate comparative quantification of total and oxidized PTP fractions. Each pool undergoes independent processing and isolation of the PTPs in each sample.

[0075]

[0070] As used herein, the term “total PTP pool” refers to the combined population of active (reduced) and inactive (oxidized) protein tyrosine phosphatases (PTPs) present in a sample. The term “oxidized PTP pool” refers to the subset of PTPs that are oxidized (inactive) in the native biological context. The relative enzymatic activity of each PTP is inferred by subtracting the signal intensity corresponding to the oxidized PTP pool (pool B) from that of the total PTP pool (pool A), thereby quantifying the enzymatically active (reduced) fraction of each PTP.

[0076]

[0071] In various embodiments, each sample pool (A) and (B) may be treated with a lysis buffer. The lysis buffers used for sample pool (A) may be distinct from the lysis buffer used for sample pool (B) . In various embodiments, sample pool (A) may be treated with lysis buffer (A), or a first lysis buffer, and sample pool (B) may be treated with lysis buffer (B), or a second lysis buffer. In particular, Pool A is treated with a lysis buffer comprising a reducing agent to converting all cysteine residues to the thiolate anion state (S-), reflecting the total PTP content, whereas Pool B is treated with a lysis buffer comprising an alkylating agent to irreversibly block reduced cysteine residues, leaving endogenously oxidized PTPs unmodified.

[0077]

[0072] In various embodiments, the lysis buffer used for sample pool (A) comprises dithiothreitol (DTT) to reduce all the PTPs within the sample into a thiolate anion state (S-).

[0078]

[0073] In various embodiments, the lysis buffer used for sample pool (B) comprises N- Ethylmaleimide (NEM) to permanently lock all the active-reduced as thiolate anion state (S-) PTPs, leaving the inactive-oxidized as sulphenic acid state (S-OH) PTPs unaltered. In various embodiments, a reducing agent such as dithiothreitol (DTT) may be subsequently added to the sample pool (B) to reduce the inactive-oxidized as sulphenic acid state (S-OH) PTPs into thiolate anion state (S-), leaving the previously locked with N-Ethylmaleimide (NEM) PTPs unaltered.

[0079]

[0074] In various embodiments, following treatment with the lysis buffers, the sample pool (A) and (B) may be sonicated and centrifuged.

[0080]

[0075] In various embodiments, after mixing with the lysis buffer, the chemical derivatization may comprise hyperoxidising the sample, or sample pools (A) and (B), and PTPs comprised therein. In various embodiments, the hyperoxidizing of the sample(s) may comprise the use and addition to the sample of a hyperoxidizing agent under conditions suitable to modify the oxidation state of the PTPs. The hyperoxidizing agent may be selected to induce irreversible hyperoxidation, such as to the sulfonic acid form (-SO3H). The hyperoxidizing agent may comprise, for example, pervanadate (a mixture of hydrogen peroxide and sodium orthovanadate), hydrogen peroxide, diamide, or other thiolreactive oxidants capable of modifying cysteine residues.

[0076] In various embodiments, the hyperoxidizing agent may be pervanadate (comprising H2O2 and Sodium Orthovanadate) and is mixed with the sample under suitable conditions to convert all PTPs into a permanently inactive sulfonic acid state (S-OaH). The hyperoxidation reaction is typically performed at room temperature (approximately 20°C to 25°C) for a period of about 15 minutes. The reaction is preferably carried out in the dark to prevent light-induced degradation of the hyperoxidizing agent. Gentle mixing or agitation may be applied during incubation to ensure uniform exposure of the proteins to the hyperoxidizing agent. After hyperoxidisation the sample(s) may be referred to as hyperoxidized samples.

[0081]

[0077] This hyperoxidation converts the thiolate anion state (S ) of cysteine into the permanently oxidized sulfonic acid state (S-O3H). In various embodiments, this conversion results in a mass increase of approximately +48 Daltons (Da) per modified cysteine residue due to the addition of three oxygen atoms to the sulfur atom. In mass spectrometry analysis, this modification is annotated as a cysteine residue bearing a +48 Da mass shift. Thus, the hyperoxidized cysteine may serve as a stable chemical tag that allows for the selective enrichment, detection / identification, and quantification of PTPs containing the modified catalytic site by parallel reaction monitoring (PRM) mass spectrometry. “Hyperoxidized cysteine" may refer to cysteine residues irreversibly oxidized to sulfonic acid (-SO3H), resulting in a +48 Da mass shift detectable by MS.

[0082]

[0078] After hyperoxidisation, the sample(s) may be subject to further sample preparation steps, prior to isolation of the PTPs via immunoprecipitation.

[0083]

[0079] In various embodiments, the chemical derivatization may be evaluated, whereby such evaluation may be performed by mass spectrometry or western blotting to confirm the hyperoxidation.

[0084]

[0080] In various embodiments, the processing step may further comprise protein digestion of the sample, or each sample pool (A) and (B) . In various embodiments, the sample, or each sample pool (A) and (B) is subjected to proteolytic digestion to generate PTP peptides. The proteolytic digestion may be performed using trypsin protease. In various embodiments, trypsin is used to achieve efficient and reproducible cleavage at lysine and arginine residues while ensuring optimal peptide length for downstream mass spectrometry analysis. The digestion reaction may be typically conducted at an appropriate temperature, such as 37°C, for a suitable incubation period, such as 12 to 16 hours, to ensure complete digestion of the proteins into peptides.

[0085]

[0081] As used herein, the term "PTP peptides" refers to peptide fragments derived from PTPs. In various embodiments, PTP peptides comprise amino acid sequences of between 9 and 29 residues in length.

[0082] In various embodiments, the PTP peptides comprise a PTP catalytic site. In various embodiments, the PTP catalytic site comprises a modification. A modified PTP catalytic site may refer to the modification of one amino acid residue, specifically cysteine residue in the motif. In various embodiments, the modification may comprise a cysteine residue in the PTP catalytic site being hyperoxidized. Thus, the proportion of active PTPs (i.e. active- reduced PTP as thiolate anion state (S-)) and inactive PTPs (i.e. inactive-oxidized PTP as sulphenic acid state (S-OH)) in the sample may be evaluated and / or quantified based on the detected, and optionally quantified, PTPs comprising a modified PTP catalytic site. For example, by subtracting the oxidized PTP quantity (Pool B) from the total PTP quantity (Pool A), the abundance of enzymatically active (reduced) PTPs may be measured.

[0086]

[0083] As used herein, the term “PTP catalytic site” refers to the catalytic site of PTPs which is highly conserved and includes a specific amino acid sequence motif centred around a critical cysteine residue. This cysteine residue is essential for the enzyme's phosphatase activity, as it forms a phospho-cysteine intermediate with the substrate's phosphate group.

[0087]

[0084] In various embodiments, the PTP peptides comprise a PTP catalytic site comprising or consisting of (i) an amino acid sequence motif HCXn, wherein X can be any amino acid residue and n is an integer of 0-6; or (ii) an amino acid sequence motif HCXXXXX[R / Q], wherein X can be any amino acid residue. In various embodiments, the PTP catalytic site comprises or consists of an amino acid sequence motif HCXnR, wherein X can be any amino acid residue and n is an integer of 0-5. In various embodiments, the PTP catalytic site comprises or consists of an amino acid sequence motif HCXnGR, wherein X can be any amino acid residue and n is an integer of 1 -4.

[0088]

[0085] In various embodiments, the cysteine residue in the PTP catalytic site is modified to increase the molecular weight of the peptide by approximately 48 Daltons (i.e. via the hyperoxidation step). In various embodiments, the cysteine residue in the catalytic site of the PTP peptide is chemically modified such that the enzymatic activity of the peptide is attenuated, and the modification increases the molecular weight by approximately 48 Daltons.

[0089]

[0086] In various embodiments, the PTP peptides may comprise a region N-terminal to the PTP catalytic site. In various embodiments, the PTP peptide includes an N-terminal flanking amino acid sequence relative to the PTP catalytic site, which may serve as a proteotypic fragment for identification and / or quantification of PTP expression or activity. The N-terminal flanking amino acid sequence may also be referred to as a PTP amino acid sequence upstream of the PTP catalytic site. As used herein, the phrase ''N-terminal flanking amino acid sequence relative to the PTP catalytic site" refers to a specific sequence of amino acids in the PTP peptide that is located upstream (or towards the N-terminus of) the amino acid sequence of the PTP catalytic site. The PTP catalytic site is positioned towards the C-terminal / terminus of the PTP peptide, and downstream relative to the N- terminal flanking amino acid sequence. Accordingly, the PTP peptide disclosed herein may comprise the PTP catalytic site and an amino acid sequence upstream of the PTP catalytic site, whereby the upstream amino acid sequence is specific and unique for each PTP to be detected, identified and distinguished from other PTPs.

[0090]

[0087] In various embodiments, the PTP peptides may comprise or consist of an amino acid sequence of at least 9 amino acids in length, and may range in length from 9-29 amino acids.

[0091]

[0088] In various embodiments, the PTP peptides comprising the PTP catalytic site and upstream amino acid sequence, and their modified (hyperoxidized) sequence may be selected from the following Table 2.

[0092]

[0089] Table 2: List of human PTP peptides and modified PTP peptides

[0093]

[0090] In various embodiments, the PTP peptides may comprise or consist of an amino acid sequence set forth in any one of SEQ ID NO: 1-47, or variants thereof, in various embodiments, a single C residue in each ot SEQ ID NO: 1-47 may be modified to increase the molecular weight of the peptide by approximately 48 Daltons. More particularly, the C residue modified is in the catalytic site and the motif HCXn(i.e. the C residue that immediately follows the H residue). For example, the PTP peptide of PTPRT comprises the modified C residue at position 14 relative to the position numbering of SEQ ID NO: 1, or at position 5 relative to the position numbering of SEQ ID NO: 2.

[0091] The term “variant”, as used herein in relation to the amino acid sequences of the PTP peptides disclosed that comprise or consist of an amino acid sequence that is at least 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91 .5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.25%, or 99.5% identical to the amino acid sequence set forth in any one of SEQ ID NO:1- 47 over their entire length. In such variants, the PTP catalytic site, may be invariable.

[0094]

[0092] In various embodiments, at least one PTP peptide may be detected for each PTP listed in Table 1. In various embodiments, two or more PTP peptides may be detected for each PTP listed in Table 1. In Table 1 , the 37 PTPs include 20 receptor-type PTPs (RPTPs), some of which (10 out of 20) possess two intracellular catalytic domains arranged in tandem: Domain 1 (D1) and Domain 2(D2). These domain-specific sequences are unique to the respective PTPs and can serve as precursor peptides in targeted MS. Therefore, some PTPs in Table 2 include two unique sequences representing the two catalytic domains of RPTPs. Detection of either sequence is sufficient to demonstrate the presence of the PTP in the sample.

[0095]

[0093] In various embodiments, the PTP peptide of PTPRT may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 1 and / or 2, or variants thereof. In various embodiments, the modified C residue is al position 14 relative to trie position numbering of SEQ ID NO: 1. In various embodiments, the modified C residue is at position 5 relative to the position numbering of SEQ ID NO: 2.

[0096]

[0094] In various embodiments, the PTP peptide of PTPRC may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 3 and / or 4, or variants thereof. In various embodiments, the modified C residue is at position 16 relative to the position numbering of SEQ ID NO: 3. In various embodiments, the modified C residue is at position 8 relative to the position numbering of SEQ ID MO: 4.

[0097]

[0095] In various embodiments, the PTP peptide of PTPRF may comprise or consist of the amino acid sequence sei forth in SEQ ID NO: 5 and / or 6, or variants thereof. In various embodiments, the modified C residue is at position 14 relative to the position numbering of SEQ ID NO: 5. In various embodiments, the modified C residue is at position 13 relative to the position numbering of SEQ ID NO: 6.

[0098]

[0096] In various embodiments, the PTP peptide of PTPN2 may comprise or consist of She amino acid sequence set forth in SEQ ID NO: 7, or variants thereof. In various embodiments, the modified C residue is at position 16 relative to the position numbering of SEQ ID NO: 7.

[0097] In various embodiments, the PTP peptide of PTPN1 may comprise or consist of the amino acid sequence set forth in SEQ IS NO: 8, or variants thereof. In various embodiments, the modified C residue is at position 16 relative to the position numbering of SEQ iD NO; 8.

[0099]

[0098] In various embodiments, the PTP peptide of PTPRA may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 9 and / or 10. or variants thereof. In various embodiments, the modified C residue is at position 14 relative to the position numbering of SEQ ID NO: 9 and / or 10

[0100]

[0099] In various embodiments, the PTP peptide of PTPPB may comprise or consist of fhe amino acid sequence set forth in SEQ ID NO: 11, or variants thereof. In various embodiments, the modified C residue is at position 11 relative to the position numbering oi SEQ ID NO; 11.

[0101]

[0100] In various embodiments, the PTP peptide of PTPRD may comprise or consist oi ihe amino acid sequence set forth in SEQ ID NO: 12 and / or 13. or variants thereof. In various embodiments, the modified C residue is at position 14 relative to the position numbering of SEQ ID NO: 12, In various embodiments, fhe modified C residue is at position 13 relative to the position numbering of SEQ ID NO: 13.

[0102]

[0101] In various embodiments, the PTP peptide of PTPRE may comprise or consist cl lhe amino acid sequence set forth in SEQ ID NO: 14 and / or 15, or variants thereof. In various embodiments, the modified C residue is at position 14 relative to the position numbering of SEQ ID NO: 14 or 15.

[0103]

[0102] In various embodiments, the PTP peptide of P I PRG may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 16, or variants thereof. In various embodiments, the modified C residue is at position 11 relative to the position numbering of SEQ ID NO: 16.

[0104]

[0103] In various embodiments, the PTP peptide of PTPRZ may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 17, or variants thereof. In various embodiments, the modified C residue ® at position 10 relative to the position numbering oi SEQ ID NO: 17.

[0105]

[0104] In various embodiments, the PTP peptide of PTPN3 may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 18, or variants thereof. In various embodiments, the modified C residue is at position 10 relative to the position numbering of SEQ ID NO: 18.

[0106]

[0105] In various embodiments, the PTP peptide of PTPRM may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 19 and / or 20, or variants thereof. In various embodiments, the modified C residue is at position 12 relative to the position numbering of SEQ ID NO: 19. In various embodiments, the modified C residue is at position 5 relative to the position numbering of SEQ ID

[0106] In various embodiments, the PTP peptide of PTPN4 may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 21, or variants thereof. In various embodiments, the modified C residue is at position 8 relative to the position numbering of SEQ ID NO: 21.

[0107]

[0107] In various embodiments, the PTP peptide of PTPN6 may comprise or consist of the ammo acid sequence set forth in SEQ ID NO: 22, or variants thereof. In various embodiments, a C residue in SEQ ID NO: 22 may be modified to increase the molecular weight of the peptide by approximately 48 Daltons. In various embodiments, the modified C residue is at position 14 relative to the position numbering of SEQ ID NO; 22.

[0108]

[0108] In various embodiments, the PTP peptide of PTPNz may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 23, or variants thereof. In various embodiments, the modified C residue is at position 22 relative to the position numbering of SEQ ID NO: 23.

[0109]

[0109] In various embodiments, the PTP peptide of PTPN9 may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 24, or variants thereof. In various embodiments, the modified C residue is at position 12 relative to the position numbering of SEQ ID NO: 24.

[0110]

[0110] In various embodiments, the PTP peptide of aTPNo may comprise or consist of lhe amino acid sequence set forth in SEQ ID NO: 25. or variants thereof. In various embodiments, the modified C residue is at position 20 relative to the position numbering of SEQ ID NO: 25.

[0111]

[0111] In various embodiments, the PTP peptide of PTPN12 may comprise or consist of the ammo acid sequence set forth in SEQ ID NO: 26. or variants thereof. In various embodiments, the modified C residue is at position 13 relative to the position numbering of SEQ ID NO: 26.

[0112]

[0112] In various embodiments, the PTP peptide of PTPN11 may comprise or consist oi the amino acid sequence set forth in SEQ ID NO: 27, or variants thereof. In various embodiments, the modified C residue a at position 14 relative to the position numbering oi SEQ ID NO: 27.

[0113]

[0113] In various embodiments, the PTP peptide of PTPRJ may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 28, or variants thereof. In various embodiments, the modified C residue is at position 12 relative to the position numbering of SEQ ID NO: 28.

[0114]

[0114] In various embodiments, the PTP peptide of PTPN13 may comprise or consist of the ammo acid sequence set forth in SEQ ID NO: 29, or variants thereof In various embodiments, the modified C residue is at position 8 relative to the position numbering of SEQ ID NO: 29.

[0115]

[0115] In various embodiments, the PTP peptide of PTPRS may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 38 and / or 31 , or variants thereof. In various embodiments, the modified C residue is a? position 14 relative to the position numbering of SEQ ID NO: 30. In various embodiments, the modified C residue is at position 13 relative to the position numbering of SEQ ID

[0116] NO: 31.

[0117]

[0116] In various embodiments, the PTP peptide of PTPRR may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 32, or variants thereof. In various embodiments, the modified C residue is at position 7 relative to the position numbering of SEQ ID NO: 32

[0118]

[0117] In various embodiments, the PTP peptide of PTPPK may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 33 and / or 34, or variants thereof. In various embodiments, the modified C residue is at position 14 relative to the position numbering of SEQ ID NO: 33. In various embodiments, the modified C residue is at position 5 relative to the position numbering of SEQ ID NO: 34.

[0119]

[0118] In various embodiments, She PTP peptide of PTPN14 may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 35, or variants thereof. In various embodiments, the modified C residue is at position 8 relative to the position numbering of SEO ID NO: 35.

[0120]

[0119] In various embodiments, the PTP peptide of PTPN21 may comprise or consist of trie amino acid sequence set forth in SEQ ID NO: 36. or variants thereof. In various embodiments, the modified

[0121] C residue is at position 19 relative to the position numbering of SEQ ID NO: 36.

[0122]

[0120] In various embodiments, the PTP peptide of P I PRO may comprise or consist of the ammo acid sequence set forth in SEQ ID NO: 37, or variants thereof. In various embodiments, the modified C residue is at position 7 relative to the position numbering of SEQ ID NO: 37.

[0123]

[0121] In various embodiments, the PTP peptide of PTPRN may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 38, or variants thereof. In various embodiments, the modified C residue ® at position 8 relative to the position numbering of SEQ ID NO: 33.

[0124]

[0122] In various embodiments, She PTP peptide of PTPN2G may comprise or consist oi the amino acid sequence set forth in SEQ ID NG: 39, or variants thereof. In various embodiments, the modified C residue is at position 11 relative to the position numbering of SEQ ID NO: 39.

[0125]

[0123] In various embodiments, the PTP peptide of PTPRU may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 40 and / or 41 , or variants thereof. In various embodiments, the modified C residue is at position 14 relative to the position numbering of SEQ ID NO: 40. In various embodiments, the modified C residue is at position 5 relative to the position numbering of SEQ ID NO: 41.

[0124] In various embodiments, the PTP peptide of PTPRN2 (PTPR2) may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 42, or variants thereof. In various embodiments, the modified C residue is at position 8 relative to the position numbering of SEO ID NO: 42

[0126]

[0125] In various embodiments, the PTP peptide of PTPN18 may comprise or consist of the ammo acid sequence set forth in SEQ ID NO: 43, or variants thereof. In various embodiments, the modified C residue is at position 13 relative to the position numbering of SEQ ID NO: 43

[0127]

[0126] In various embodiments, the PTP peptide of PTPN23 may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 44, or variants thereof. In various embodiments, the modified C residue is at position 23 relative to the position nuntoering oi SEQ ID NO; 44.

[0128]

[0127] In various embodiments, the PTP peptide of PTPRH may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 45, or variants thereof. In various embodiments, the modified C residue is at position 16 relative to the position numbering of SEQ ID NO: 45.

[0129]

[0128] In various embodiments, the PTP peptide of PTPRQ may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 46, or variants thereof. In various embodiments, the modified C residue is at position 11 relative to the position numbering of SEQ IQ NO: 46.

[0130]

[0129] In various embodiments, the PTP peptide of PTPN22 may comprise or consist of the ammo acid sequence set forth in SEQ ID NO: 47, or variants thereof. In various embodiments, the modified C residue is at position 14 relative to the position numbering of SEQ ID NO: 47.

[0131]

[0130] Following protein digestion, the resulting sample mixtures may contain residual salts, detergents, chaotropic agents, and small molecules originating from earlier processing steps. In various embodiments, the processing step may further comprise peptide purification of the sample, or each sample pool (A) and (B). The peptide purification step, may also be referred to as peptide clean-up, and may be performed to remove such contaminants and prepare the sample(s) for subsequent immunoprecipitation via antibody enrichment and LC-MS / MS analysis. The peptide purification may be performed by desalting methods including, but not limited to, solid-phase extraction (SPE) using reversed-phase C18 cartridges or plates, spin column desalting, ultrafiltration, or dialysis. In certain embodiments, reversed-phase SPE is employed to selectively retain peptides while allowing removal of small molecule contaminants by washing. Elution is typically performed with an organic solvent such as acetonitrile with acid (e.g., 0.1% formic acid), yielding purified peptides in a volatile buffer suitable for immunoprecipitation.

[0132]

[0131] In various embodiments, the hyperoxidized cysteine residues (sulfonic acid state, S-O3H) in the PTP peptides remain chemically stable and intact throughout digestion and peptide purification, thereby preserving the modification as a stable mass tag for downstream enrichment and detection. The peptide purification step enhances the specificity, sensitivity, and reproducibility of subsequent immunoprecipitation and mass spectrometry analysis.

[0133]

[0132] In various embodiments, the step of isolating or enriching the RTFs (i.e. FTP peptides) in the processed sample, or samples, may comprise antibody-based immunoprecipitation specific for FTP peptides containing hyperoxidized cysteine residues in the sulfonic acid (S-O3H) state. In various embodiments, the immunoprecipitation may be performed after protein digestion and peptide purification, wherein PTP peptides containing sulfonic acid-modified cysteine residues are selectively captured by binding to immobilized antibodies.

[0134]

[0133] In various embodiments, the antibody-based immunoprecipitation employs an antibody that is specific for cysteine sulfonic acid-modified PTP peptides, hereinafter referred to as an ox-PTP antibody. The ox-PTP antibody selectively and specifically binds to peptides containing cysteine residues that have been hyperoxidized to the inactive sulfonic acid (S-O3H) state, thereby enriching for peptides derived from the catalytic domains of PTPs that underwent hyperoxidation during sample preparation. In various embodiments, the ox-PTP antibody may be crosslinked to a solid support matrix, such as agarose, magnetic beads, or other chromatography media, to enable efficient immunoprecipitation and washing steps while minimizing antibody leaching during peptide elution.

[0135]

[0134] In various embodiments, the antibody-based immunoprecipitation may be evaluated for the isolation and enrichment of the PTP peptides. The evaluation of enrichment refers to assessing the selectivity, efficiency, and purity of the immunoprecipitation process in isolating PTP peptides containing hyperoxidized cysteine residues derived from PTP catalytic domains. Evaluation may include comparing the peptide composition of pre-immunoprecipitation input samples, negative controls, and post-immunoprecipitation eluates using analytical methods such as mass spectrometry (MS)if intact / non-digested proteins are used for the enrichment, or immunodetection assays employing antibodies specific for sulfonic acid-modified peptides. Such assessments confirm the selective recovery of the target PTP peptides while minimizing nonspecific background binding. In various embodiments, successful enrichment may be demonstrated by MS analysis.

[0136]

[0135] After antibody-based immunoprecipitation, the prepared sample(s) (i.e. immunoprecipitated PTP peptide fraction), may be analysed using liquid chromatography tandem mass spectrometry (LC- MS / MS) operated in scheduled parallel reaction monitoring (PRM) mode.

[0137]

[0136] In various embodiments, the tandem mass spectrometry comprises a mass spectrometry scheduled Parallel Reaction Monitoring (PRM) acquisition method. In various embodiments, the tandem mass spectrometry comprises a first mass spectrometry stage (MS1 ) and a second mass spectrometry stage (MS2), wherein the MS2 is in the sPRM mode.

[0137] The LC-MS / MS comprises first separating the PTP peptides by reverse-phase chromatography. This separation improves peptide resolution and facilitates efficient ionization. As peptides elute from the chromatography column, they are ionized, commonly via electrospray ionization (ESI), and enter the mass spectrometer. Each ionized peptide is referred to as a precursor ion, characterized by a specific mass-to-charge ratio (m / z) and charge state. In various embodiments, each detected and quantified precursor ion directly corresponds to a specific PTP peptide, allowing inference of the expression level and / or enzymatic activity of the originating PTP protein.

[0138]

[0138] In various embodiments, the LC-MS / MS analysis may comprise reverse-phased chromatography. In reverse-phased chromatography, peptide separation may be achieved based on hydrophobic interactions using a C18 column under a gradient of increasing organic solvent concentration (e.g., acetonitrile or methanol with 0.1 % formic acid). Reverse-phase separation may improve peak capacity, retention time reproducibility, and compatibility with electrospray ionization (ESI), making it a preferred method for peptide analysis in LC-MS / MS workflow.

[0139]

[0139] In various embodiments, the LC-MS / MS analysis may comprise a Ultra-High Performance Liquid Chromatography (UHPLC) system. UHPLC may enable high-pressure operation and enhanced chromatographic resolution using columns packed with sub-2 pm particles. The UHPLC system may allow rapid and reproducible separation of complex peptide mixtures derived from PTPs with improved peak definition and sample throughput.

[0140]

[0140] In various embodiments, the liquid chromatography (LC) may comprise a gradient of at least 75 minutes. In particular, the liquid chromatography separation may be performed using a gradient elution over approximately 75 minutes, optionally including equilibration and wash steps. The gradient may be optimized to achieve maximum separation of co-eluting peptides and minimize matrix effects, thereby enhancing sensitivity in downstream MS detection.

[0141]

[0141] In various embodiments, the LC-MS / MS analysis may be performed using a tribrid mass spectrometer, such as an instrument combining quadrupole, ion trap, and Orbitrap analysers. Tribrid systems allow for flexible fragmentation strategies (e.g., HCD, CID, ETD) and high-resolution detection of both precursor and fragment ions, facilitating improved identification and characterization of PTP peptides, including post-translationally modified species.

[0142]

[0142] As used herein, the term “PRM" refers to Parallel Reaction Monitoring, a targeted mass spectrometry technique used for the selective and quantitative analysis of specific peptides within complex biological samples. In PRM, a quadrupole mass filter is used to selectively isolate a precursor ion of interest, which is then subjected to fragmentation in a collision cell (e.g., higher-energy collisional dissociation, HCD). The resulting fragment ions are detected using a high-resolution and accurate-mass (HRAM) analyzer, such as an Orbitrap or time-of-flight (TOF) detector. Unlike Multiple Reaction Monitoring (MRM), which monitors predefined fragment ion transitions, PRM collects full MS / MS spectra for each precursor, allowing for post-acquisition selection of fragment ions for quantification and enhanced specificity. Scheduled Parallel Reaction Monitoring (sPRM) is an advanced implementation of PRM in which precursor ion selection and MS / MS acquisition are restricted to predefined retention time windows, based on prior knowledge of peptide elution times. In sPRM, the quadrupole mass filter selectively isolates target precursor ions only during their expected elution periods, as defined in a scheduled acquisition list. This approach increases the number of peptides that can be monitored per run, improves dwell time and sensitivity, and optimizes instrument duty cycle. By limiting acquisition to specific time windows, sPRM enhances quantitative reproducibility, detection sensitivity, and throughput, making it particularly suitable for high-complexity proteomic studies and multiplexed targeted protein quantification.

[0143]

[0143] LC-MS / MS acquisition in sPRM mode includes several parameters including but not limited to: Retention Time parameter (Information on the exact time each target peptide elutes from the chromatographic column, used for identifying and confirming peptides based on their expected retention times); Scheduled Acquisition Windows (Details about the predefined time windows during which the mass spectrometer acquires data for each target peptide); Precursor Ion Information (Data on the selected precursor ions, including their mass-to-charge (m / z) ratios and the specific time windows during which they were monitored).

[0144]

[0144] In various embodiments, the scheduled PRM mode may comprise parameters of pre-defined and selected mass-to-charge ratios (m / z) and retention time (RT) windows for the PTPs, preferably for PTP peptides.

[0145]

[0145] In various embodiments, the mass-to-charge ratios (m / z) for both doubly charged (+2) and triply charged (+3) ion states, and / or optionally a quadruply charged (+4) ion state, of the PTP peptides may be used for the scheduled PRM in the LC-MS / MS analysis. As shown in the working examples disclosed herein, PTP peptides are obtained in +2 and / or +3 and / or +4 charge states, thus ensuring comprehensive detection and identification of PTPs in the sample. Inclusion of multiple charge states of the PTP peptides may increase the likelihood of successful detection across varying sample conditions and provide redundancy for peptide identification and quantification. In various embodiments, the scheduled PRM method may be configured to selectively isolate both charge states during their respective retention time windows, thereby enhancing detection sensitivity and specificity. The selection of charge states may also be optimized based on PTP peptide length, amino acid composition, and the presence of basic residues (e.g., lysine, arginine, histidine), which influence the ionization pattern under electrospray ionization (ESI) conditions.

[0146]

[0146] In various embodiments, the retention time (RT) may be configured to prevent noise peaks from near - isobaric peptide ions, and to increase the number of transitions that can be obtained for each peptide, enhancing quantification. As used herein, the term "isobaric peptide ions" refers to peptide ions that exhibit the same mass-to-charge ratio (m / z) as one or more peptides of interest, such as those peptides derived from protein tyrosine phosphatases (PTPs), but that differ in primary amino acid sequence and / or chemical composition. These isobaric peptides may interfere with precursor ion detection in mass spectrometry; however, they can be resolved based on distinct fragmentation patterns in tandem mass spectrometric (MS / MS) analysis.

[0147]

[0147] Each retention time (RT) window may be characterized by the precursor ion m / z value corresponding to the peptide mass incorporating the hyperoxidized cysteine modification (+48 Da), typically ranging from m / z 400 to 1300, depending on the molecular weight of the peptide and its charge state, which may be +2 or +3 or +4. For each precursor ion, a retention time window is defined to restrict acquisition to the period during which the peptide elutes from the chromatography column. Retention time start points may range from 10 minutes to 60 minutes following chromatographic injection, with retention time windows typically spanning 2 to 10 minutes in width from a start to an end time. In various embodiments, retention time windows may be optimized or narrowed based on empirical retention time alignment, retention time normalization using internal standards, or historical reference data obtained from prior analyses of PTP peptides. This scheduling allows efficient monitoring of multiple PTP peptides with high specificity and sensitivity while maximizing duty cycle and minimizing acquisition redundancy.

[0148]

[0148] In various embodiments, one or more pre-defined precursor ions corresponding to PTP peptides derived from protein tyrosine phosphatases (PTPs) may be selectively isolated, fragmented, and their product ion spectra are detected within defined retention time windows, enabling highly sensitive and specific identification of PTP isoforms.

[0149]

[0149] In various embodiments, the parameters of the LC-MS / MS operated in scheduled PRM mode is configured to detect and distinguish between human PTPs based on the PTP amino acid sequence upstream of the PTP catalytic site comprised in the PTP peptides.

[0150]

[0150] In various embodiments, the parameters of the LC-MS / MS may comprise one or more of the following:

[0151] (I) selected PTP peptide sequences corresponding to each human PTP, preferably the PTP peptide comprise the PTP amino acid sequence upstream of the PTP catalytic site;

[0152] (ii) modification of the PTP peptide sequence (i), such as a modified cysteine (C) residue in the PTP catalytic site to add 48 Daltons (Da) to the molecular weight of the peptide (i));

[0153] (iii) charge state (z) for each PTP peptide sequence (i);

[0154] (iv) precursor m / z of each PTP peptide sequence (I);

[0155] (v) retention time of each PTP peptide sequence (I);

[0156] (vi) adjusted retention time; and / or

[0157] (vii) minimum start time and minimum end time related to the RT.

[0158]

[0151] In various embodiments, the parameters of the LC-MS / MS may comprise all of (i)-(vii).

[0152] In various embodiments, the retention time with a start and end (mins) for each PTP may be as defined in Table 3 below, such that the method is capable of identifying and distinguishing between human RTFs listed in Table 1 in a mple. It will be appreciated that the retention times provided in the accompanying table (including start and end times) are approximate values and are to be interpreted as encompassing a degree of variation, for example the retention time of PTPN1 may be at 34min of chromatographic gradient ± 0.5 min. The 10min window accounts for the potential variability in LC performance.

[0159]

[0153] Table 3: Retention times for all 37 human FTP peptides (times are in minutes, and 2 / 3 means that both charge states 2 and 3 apply, or 3 / 4 means that both charge states 3 and 4 apply).

[0160]

[0154] In various embodiments, the parameters of the analysis operating in scheduled PRM mode of the human PTPs, are defined in Table 5 below, such that the method is capable of identifying and distinguishing between 37 human PTPs listed in Table 1 in a sample.

[0161]

[0155] Data output from sPRM mode measurements includes high-resolution MS / MS spectra acquired for specific precursor ions within predefined retention time windows. These MS / MS spectra contain fragment ion mass-to-charge (m / z) ratios and corresponding signal intensities, which are used to confirm peptide identity and quantify peptide abundance. Quantification is performed based on extracted ion chromatograms (XICs) of selected fragment ions, with the resulting chromatographic peak areas representing the relative concentration of the peptides in the sample. These fragment ions are selected post-acquisition for each precursor, allowing high specificity and reproducibility. Additional data outputs include the retention times of each detected peptide, signal intensities of selected fragment ions, and computed quantitative values (e.g., peak area or peak area ratios). Quality control metrics such as signal-to-noise ratio, peak shape, and retention time reproducibility are also obtained to assess the reliability and analytical performance of the sPRM analysis.

[0162]

[0156] In various embodiments, the detection step may comprise detecting, and optionally quantifying, PTP peptides in the sample. In various embodiments, the detection step further comprises distinguishing between each PTP identified in the sample.

[0163]

[0157] In various embodiments, a precursor ion spectrum and / or a fragment ion spectrum is obtained from the LC-MS / MS analysis to detect, and optionally quantify, the expression level and / or enzymatic activity of PTPs in the sample. As used herein, the term “precursor ion spectrum” refers to a spectrum that displays the mass-to-charge (m / z) ratios of the precursor ions selected for fragmentation. In various embodiments, the PTP peptides are in an ionized form and may be termed as precursor ions. The precursor ion corresponds to the PTP peptide in a defined charge state (e.g., +2 or +3), and is used as the target ion in the scheduled PRM acquisition method. As used herein, the term “fragment ion spectrum" refers to a spectrum that is generated after the selected precursor ions are fragmented in the mass spectrometer. It shows the m / z ratios and intensities of the resulting fragment ions. This spectrum confirms the identity of the peptides by matching the observed fragment ions to theoretical or reference spectra and for quantifying the peptides based on the intensity of the fragment ions.

[0164]

[0158] The identification of specific FTP peptides of interest may be performed based on their characteristic precursor ion m / z values and MS / MS fragmentation patterns, including the m / z values of the resulting fragment ions.

[0165]

[0159] In various embodiments, the outcome of the LC-MS / MS analysis will be the detection, and optional quantification, of the expression level and / or enzymatic activity of the PTPs in the sample.

[0166]

[0160] In various embodiments, the detection step further comprises distinguishing between each PTP identified in the sample. In this regard, the expression and / or activity level of individual or set of PTPs may be determined in a sample.

[0167]

[0161] As used herein, the term "expression level of PTPs" refers to the abundance of PTPs present in the sample, determined by detecting one or more peptides derived from the PTPs using scheduled parallel reaction monitoring (PRM) in an LC-MS / MS analysis. Quantification may be based on chromatographic peak area, fragment ion intensity, or precursor ion signal corresponding to uniquely identifiable PTP peptides. The expression level may be measured in absolute or relative terms and may be compared to a reference or control sample to assess differential expression. In various embodiments, the expression levels of the PTPs may be quantified from a processed sample (e.g. Pool A) representing the total PTP abundance (active + inactive PTPs).

[0168]

[0162] As used herein, the term "enzymatic activity of PTPs" refers to the functional status of PTPs based on the oxidation state of their catalytic cysteine residues. Active PTPs exist in a reduced thiolate anion state (S“), while inactive PTPs exist in an oxidized sulfenic (S-OH), sulfinic (S-02H)or sulfonic acid (S-O3H) state.

[0169]

[0163] In various embodiments, the enzymatic activity is assessed by chemically distinguishing between oxidized (inactive) and reduced (active) forms of PTPs, wherein the catalytic cysteine is differentially derivatized, and the resulting peptides are detected and quantified based on the presence or absence of oxidation-specific modifications (e.g., +48 Da for hyperoxidation). The abundance of modified and unmodified peptides enables quantitative inference of the functional state of PTPs in the sample.

[0170]

[0164] In various embodiments, the enzymatic activity may be determined by selectively derivatizing, isolating, and analyzing the oxidized and reduced forms of PTPs in separate sample pools (A) and (B). Following conversion of PTPs to a uniform inactive sulfonic acid state (S-O3H), LC-MS / MS identification and quantification of PTP peptides containing the modified catalytic site allows calculation of the relative or absolute abundance of active and inactive PTPs in a sample. The difference between total PTPs and oxidized PTPs corresponds to the proportion of active (functionally reduced) PTPs in the sample. Thus, the proportion of active PTPs (i.e. active-reduced PTP as thiolate anion state (S-)) and inactive PTPs (i.e. inactive-oxidized PTP as sulphenic acid state (S-OH)) in the sample may be evaluated and / or quantified based on the detected, and optionally quantified, PTP peptides comprising a modified PTP catalytic site.

[0171]

[0165] In various embodiments, the expression level of each protein tyrosine phosphatase (PTP) is quantified from LC-MS / MS analysis of: (i) a total PTP pool comprising both enzymatically active (reduced) and inactive (oxidized) forms; and (ii) an oxidized PTP pool, wherein only the inactive, oxidation-inhibited forms of the PTPs are detected, and the enzymatic activity level of each PTP is determined by subtracting the quantified oxidized PTP fraction from the total PTP fraction. In this regard, the oxidized PTPs serve as a proxy for the inactive PTP population, while the difference between total and oxidized PTP levels corresponds to the non-oxidized, catalytically active PTPs. This differential quantification enables the simultaneous assessment of both total protein expression and functional enzymatic activity states for each PTP, and more specifically, each of the 37 human PTPs, within a single biological sample.

[0172]

[0166] In various embodiments, the detection step may comprise detecting, and optionally quantifying, the expression level of active and inactive PTPs by the detection, and optional quantification, of PTP peptides. In various embodiments, the method allows for both identification and quantification of the expression level and enzymatic activity of protein tyrosine phosphatases (PTPs) in a sample.

[0173]

[0167] In various embodiments, the expression level and / or enzymatic activity of individual or set of PTPs detected, and optionally quantified, in the sample, is compared to the expression level and / or enzymatic activity of the individual or set of PTPs detected, and optionally quantified, in a control or comparative sample. Accordingly, the method enables simultaneous determination of both total PTP protein expression and enzymatic activity across the entire human PTP family from a single sample.

[0174]

[0168] In various embodiments, the expression level and / or enzymatic activity in the sample analysed may be compared to the expression level and / or enzymatic activity of a comparative or control sample to assess differential expression and / or activity.

[0175]

[0169] As used herein, the term “control sample” or “comparative sample” refers to any appropriate positive or negative control for a given test sample, in keeping with standard laboratory methods. For example, the control or comparative sample may be a sample obtained from a healthy individual or ceii sample known to be free from a disease that is to be detected, or alternatively from a source known to have a specific disease or display a phenotype associated with a specific disease or disorder. The control sample may be from a particular ceii or tissue type. The control or comparative sample may be a sample that has or has not been exposed to a drug or treatment regimen or a PTP agonist'antagonist, whereas the test sample may have the same or opposite treatment status as the control. The comparative or control sample may be obtained from the same source or subject as the test sample at a different time during a treatmerit regimen. The comparative or control sample may have a known activity up-regulation or down-regulation for one or more specific PTPs, for example may be a sample from a cel I known to have a mutation for a specific PTP or known to be transgenically expressing a specific PTP. The control or comparative sample may be treated to lyse cells contained in the sample. The control or comparative sample may be treated with a phosphatase inhibitor. The term “comparative sample" may be used interchangeably with “reference sample”. The term “reference sample,” “reference cell,” “reference tissue,” “control sample,” “control cell,” or “control tissue,” as used herein, refers to a sample, cell, tissue, standard, or level that is used for comparison purposes. A reference sample, reference cell, reference tissue, control sample, control cell, control tissue, may be obtained from a healthy and / or non-diseased individual, or a non-healthy and / or diseased individual.

[0176]

[0170] In various embodiments, the expression level and / or enzymatic activity in the sample analysed may be compared to an reference expression level and / or enzymatic activity to assess differential expression and / or activity. As used herein, the term “reference expression levels and / or enzymatic activity” refers to baseline or control data against which results of the method (i e PTPs identified and quantified) are compared.

[0177]

[0171] In various embodiments, the reference expression level and / or enzymatic activity may be derived from a comparative sample or control sample obtained from a subject or an individual that is not the subject, or an online medical database. The detection, and optional quantification, of the expression and / or activity level of individual or set of PTPs in a sample may be used for disease detection, diagnosis and / or prognosis.

[0178]

[0172] In various embodiments, each reference expression level and / or enzymatic activity is specific for a disease or condition associated with one or more PTPs, and / or is predictive of the presence or likelihood of such a disease or condition associated with one or more PTPs in said subject.

[0179]

[0173] In various embodiments, the method disclosed herein may be used in a method for the diagnosis, or prognosis or monitoring of a disease, based on the expression and / or activity levels of PTPs identified in a sample obtained from a subject.

[0180]

[0174] In the context of the present application, “diagnosis” and “diagnosing” generally include a determination of a subject's susceptibility to a disease or disorder, a determination as to whether a subject is presently affected by a disease or disorder, a prognosis of a subject affected by a disease or disorder, and therametrics (e g. monitoring a subject’s condition to provide information as to the effect or efficacy of therapy). The terms “prognosis” or “prognose" refer to the act or art of foretelling the course of a disease. Additionally, the terms refer to the prospect of survival recovery from a disease as anticipated from the usual course of that disease or indicated by special features of the individual case. Further, the terms refer to the art or act of identifying a disease from its signs and symptoms.

[0181]

[0175] In various embodiments, the sample may ba a biological sample that has been obtained from a subject, and the expression level and / or enzymatic activity of PTPs detected, and optionally quantified, in the biological sample is compared with reference expression levels and / or enzymatic activity of PTPs associated with a disease or condition associated with one or more PTPs. This reference data may serve as a standard for normal or known conditions / diseases associated with PTPs, allowing for the assessment of deviations or changes or similarities that may indicate the subject has, or is likely to have, a disease or other condition associated with one or more PTPs, or does not have, or is not likely to have, a disease or other condition associated with one or more PTPs.

[0182]

[0176] As used herein, the term "subject" refer to all mammals, including humans. Examples of subjects include humans, cows, dogs, cats, horses, goats, sheep, pigs, and rabbits, and may be used interchangeably with “patient" or “individual" The subject may refer to a warm-blooded animal, preferably a mammal, more preferably a human. Said subject may be awaiting or receiving medical care or is or will become the subject of a medical procedure or is being monitored for the development of any condition or disease associated with PTPs, or an aberrant expression and / or activity level of PTP. Subjects include those already being afflicted by condition or disease associated with PTPs as well as subjects susceptible to a condition or disease associated with PTPs or for whom a condition or disease associated with PTPs should be prevented or delayed.

[0183]

[0177] As used herein, the term “disease or condition associated with one or more PTPs” refers to any disease or condition that is known to be associated with aberrant expression and / or activity level of one or more PTPs (either a single PTP or a combination of PTPs), and the role of the one or more PTP in various cellular processes, including cell growth, differentiation, metabolism, and immune response. The disease or condition associated with one or more PTPs may include cancers, metabolic disorders, autoimmune diseases, neurological disorders, infectious diseases, cardiovascular diseases, developmental disorders, and inflammatory diseases.

[0184]

[0178] As used herein, the term “aberrant expression and / or activity level" of a protein (i e PTP protein) refers to any deviation from the normal, physiological levels of expression or enzymatic activity of a PTP within a biological system. This can include both overexpression and underexpression, as well as increased or decreased enzymatic activity relative to reference levels.

[0185]

[0179] In various embodiments, the disease or condition associated with one or more PTPs is cancer. In various embodiments, the cancer may be selected from: Breast Cancer, where overexpression or mutations in PTPs such as PTPN1 and PTPN11 (SHP2) can drive oncogenic signaling; Colon Cancer, where mutations in PTPRT and PTPN12 are linked to tumorigenesis; Leukemia, where abnormal activity of PTPN11 (SHP2) can lead to various leukemias, including juvenile myelomonocytic leukemia; or Lung Cancer, where PTPRD deletions or mutations are often found in lung cancer.

[0186]

[0180] In various embodiments, the disease or condition associated with one or more PTPs is a metabolic disorder. In various embodiments, the metabolic disorder may be selected from: Diabetes, where PTPN1 (PTP1 B) negatively regulates insulin signaling, and its overactivity is associated with insulin resistance and type 2 diabetes; Obesity, where elevated PTPN1 activity is also implicated in the development of obesity due to its role in metabolic signaling pathways; and progression of Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD).

[0187]

[0181] In various embodiments, the disease or condition associated with one or more PTPs is a autoimmune disease. In various embodiments, the autoimmune disease may be selected from: Type 1 Diabetes, where variants in PTPN22 are associated with an increased risk of type 1 diabetes; Rheumatoid Arthritis, where PTPN22 mutations are also linked to rheumatoid arthritis; and Systemic Lupus Erythematosus, where abnormalities in PTPN22 can contribute to lupus pathogenesis.

[0188]

[0182] In various embodiments, the disease or condition associated with one or more PTPs is a neurological disorder. In various embodiments, the neurological disorder may be selected from: Alzheimer's Disease, where dysregulation of PTPN1 (PTP1 B) has been implicated in the pathophysiology of Alzheimer's disease; and Schizophrenia, where mutations or deletions in PTPRD are associated with an increased risk of schizophrenia.

[0189]

[0183] In various embodiments, the disease or condition associated with one or more PTPs is an infectious disease. In various embodiments, the infectious disease may be Tuberculosis, where altered activity of certain PTPs can impact the host immune response to Mycobacterium tuberculosis.

[0190]

[0184] In various embodiments, the disease or condition associated with one or more PTPs is a cardiovascular disease. In various embodiments, the cardiovascular disease may be Atherosclerosis, where PTP1 B has been implicated in the development of atherosclerosis due to its role in vascular inflammation and metabolism.

[0191]

[0185] In various embodiments, the disease or condition associated with one or more PTPs is a developmental disorder. In various embodiments, the developmental disorder may be Noonan Syndrome, where mutations in PTPN1 1 (SHP2) are a common cause of Noonan syndrome, a disorder characterized by abnormal development in various parts of the body.

[0192]

[0186] In various embodiments, the disease or condition associated with one or more PTPs is an inflammatory disease. In various embodiments, the inflammatory disease may be Inflammatory Bowel Disease (IBD), where abnormal expression of certain PTPs can contribute to the pathogenesis of IBD.

[0193]

[0187] In various embodiments, the method disclosed herein may be used in a method for assisting in determining a subject's risk for developing a disease or condition associated with one or more PTPs through detecting, and optionally quantifying, the expression level and / or enzymatic activity of protein tyrosine phosphatases (PTPs) in a biological sample obtained from the subject.

[0194]

[0188] In this regard, the detection and / or quantification of PTP expression or activity in the biological sample may be indicative of the subject's current physiological state and may reflect deviations from established reference values associated with healthy or diseased states. In various embodiments, increased or decreased levels of expression and / or enzymatic activity of specific PTPs are correlated with an increased or decreased risk of developing a particular disease or condition, such as cancer, metabolic disorders, autoimmune diseases, neurological disorders, cardiovascular diseases, infectious diseases, developmental disorders, or inflammatory diseases.

[0195]

[0189] In various embodiments, the expression level and / or enzymatic activity of the one or more PTPs may be compared to one or more reference expression levels or activity profiles that are known to be associated with a defined physiological or pathological state. Based on this comparison, the subject may be categorized as being at normal, elevated, or reduced risk for developing the associated disease or condition.

[0196]

[0190] In various embodiments, the method of assessing may facilitate early risk stratification, prognosis, or monitoring of the subject’s health status, particularly where early intervention or surveillance is clinically beneficial. The method may further be used in conjunction with clinical decision-making tools or personalised medicine approaches to tailor preventive or therapeutic strategies based on the subject’s specific PTP expression or activity profile.

[0197]

[0191] In various embodiments, the method of assessing may be implemented as part of a diagnostic workflow, in combination with additional biomarkers or clinical indicators, to enhance predictive accuracy. The method may also be employed in population screening, clinical trials, or epidemiological studies aimed at understanding the role of PTP dysregulation in disease susceptibility.

[0198]

[0192] In various embodiments, the method disclosed herein may be used in a method for predicting a subject’s response to a treatment for a disease or condition associated with one or more protein tyrosine phosphatases (PTPs). In particular, the method disclosed herein and detected / quantified expression and / or activity levels of PTPs in a sample, may serve as a predictive biomarker-based approach to guide treatment decisions in a subject.

[0193] In various embodiments, the expression level and / or enzymatic activity of one or more PTPs detected may be evaluated in relation to reference profiles that correlate with known treatment responses. For example, subjects exhibiting a particular level or pattern of PTP dysregulation may be predicted to respond favorably or unfavorably to a given therapy, such as a PTP inhibitor, immune checkpoint inhibitor, chemotherapeutic agent, or metabolic modulator. The predictive value of the method lies in its ability to stratify patients based on molecular features prior to initiating treatment.

[0199]

[0194] In various embodiments, the method for predicting may comprise identifying responders or non-responders to a targeted therapy by linking PTP expression or activity levels to treatment outcomes. For example, elevated baseline PTPN1 (PTP1 B) activity may predict resistance to insulinsensitizing agents in metabolic disorders, whereas high PTPN11 expression in tumors may predict responsiveness to PTPN11 inhibitors or PTPN1 1 -adjunct immunotherapies. Such stratification informs the likelihood of therapeutic efficacy and facilitates tailored treatment regimens.

[0200]

[0195] In various embodiments, the method for predicting may further comprise selecting a treatment regimen based on the subject’s PTP profile For instance, if a subject's PTP activity profile suggests low probability of response or elevated risk of adverse effects, an alternative therapeutic strategy may be selected. Conversely, subjects whose PTP activity profile match a favorable predictive signature may be prioritized for therapy initiation or inclusion in treatment cohorts or clinical trials.

[0201]

[0196] In various embodiments, the method may form part of a companion diagnostic platform used to determine eligibility for PTP-targeted therapeutics or for combination therapies involving immunomodulatory agents, kinase inhibitors, or metabolic regulators. The output of the method may be provided in the form of a predictive report or stratification score. Accordingly, the method provides a clinically actionable tool for predicting treatment response in diseases or conditions associated with one or more PTPs, supporting precision medicine, therapy selection, and optimized patient management.

[0202]

[0197] In various embodiments, the present invention further provides a method for selecting a suitable treatment regimen in a subject with a disease or condition associated with one or more PTPs, the method comprising: predicting the responsiveness of the subject to treatment by the predicting method disclosed herein; and selecting a treatment regimen for administering to the subject.

[0203]

[0198] The term "treatment regimen" as used herein refers a treatment regimen for a disease or condition associated with one or more PTPs, the selection of which may be indicated by the methods or kits of the invention. The treatment regimen comprises provision to the subject of a PTP-targeting agent, such as a PTP inhibitor or PTP agonist, depending on the nature of the PTP dysregulation identified. Examples of PTP modulators that may be employed in such treatment regimens include, but are not limited to, small molecule inhibitors of PTPN1 or PTPN11 (PTP1 B or SHP2), or agonists that enhance the activity of immunomodulatory PTPs such as PTPN2 or PTPN22. Suitably, the PTP- targeting agent may be provided as the only (or primary) therapeutic agent in the treatment regimen. Details of treatment regimens using such PTP modulators will be known to those skilled in the art, and known regimens of this type may be used in accordance with the present invention. As referred to above, in suitable embodiments, a treatment regimen may further comprise the provision of additional agents such as chemotherapeutics, immunotherapies (e.g., checkpoint inhibitors), or targeted therapies (e.g., kinase inhibitors), in combination with the PTP-targeting agent.

[0204]

[0199] As used herein, the terms "treating" and "treatment" refer to reduction in severity and / or frequency of symptoms, elimination of symptoms and / or underlying cause, prevention of the occurrence of symptoms and / or their underlying cause, and improvement or remediation of damage. As used herein, the term “preventing” refers to the prophylactic or preventative measures that prevent and / or slow the development of a targeted pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented and those in whom reoccurrence of the disorder needs to be prevented. In various embodiments, the terms “treating", “ameliorating”, “delaying” or “preventing”, as used herein refer to achieving one or more of the following in the subject: (a) reducing the severity of a given condition; (b) limiting or preventing the development of a condition; (c) removing a given condition; (d) limiting or preventing the recurrence of a given condition; (e) alleviation of the condition and / or its symptoms; and (f) delay the onset of a condition. Any one or more of these effects may be achieved in a subject who previously had or currently has or is suspected to develop a condition or disease associated with PTPs.

[0205]

[0200] In various embodiments, the method for detecting, and optionally quantifying, the expression level and / or enzymatic activity of one or more PTPs in a biological sample may be used in a method for monitoring treatment efficacy in a subject undergoing treatment for a disease or condition associated with one or more PTPs. The method allows dynamic assessment of molecular responses to treatment by tracking changes in PTP biomarkers over the course of therapeutic intervention.

[0206]

[0201] In various embodiments, one or more biological samples may be obtained from the subject at different time points before, during, or after treatment, and the expression and / or activity levels of the PTPs are measured and compared to reference values or to prior time points from the same subject. Alterations in PTP expression or activity profiles over time may serve as indicators of therapeutic response, disease progression, or relapse. For example, a reduction in PTP activity that was previously elevated in disease states may reflect positive treatment response, whereas persistently elevated or increasing PTP activity may indicate incomplete target inhibition, therapeutic resistance, or disease recurrence.

[0207]

[0202] In various embodiments, the method enables assessment of response to targeted therapeutic interventions that modulate PTP function, including administration of PTP inhibitors (e.g., small molecules directed against PTPN1 / PTP1 B, SHP2 / PTPN11 , or PTPN2) or PTP agonists (e.g., compounds enhancing immunoregulatory PTPs such as PTPN22). A decline in aberrant PTP activity following administration of a PTP inhibitor may indicate effective target engagement and pharmacodynamic response, while persistent or rising activity despite treatment may suggest suboptimal efficacy or emergence of drug resistance. Conversely, in applications such as immunotherapy, an increase in PTP activity driven by a PTP agonist may be associated with enhanced immune regulation or restoration of immune signalling pathways, correlating with improved clinical outcomes.

[0208]

[0203] In various embodiments, the method for monitoring the treatment efficacy, may comprise administering the treatment to the subject; obtaining a sample from the subject; and detecting, and optionally quantifying, the expression level and / or enzymatic activity of one or more PTPs in a biological sample using the method disclosed herein, wherein the expression level and / or enzymatic activity of one or more PTPs in the sample is indicative of the efficacy of the, optionally these steps may be repeated two or more times within a time-frame and the detection at each time point is compared against each other to assess the progression of the disease or condition and efficacy of the treatment.

[0209]

[0204] In various embodiments, the method disclosed herein may be used in a method for developing or identifying therapeutic agents capable of modifying the expression and / or activity level of PTPs in a subject, the method comprising: measuring the expression level and / or enzymatic activity of PTPs in a first biological sample obtained from the subject using the method disclosed herein; administering a candidate therapeutic agent to the subject; measuring the expression level and / or enzymatic activity of PTPs in a second biological sample obtained from the subject using the method disclosed herein, following the administration of the candidate agent; and characterizing the candidate therapeutic agent as capable of modifying the activity of PTPs, if the subject's measured expression level and / or enzymatic activity of PTPs increases or decreases following administration of the candidate therapeutic agent.

[0210]

[0205] The term “candidate therapeutic agent" refers to an agent for which there is some evidence of potential therapeutic efficacy. The term candidate compound refers to a compound that may or may not have been previously evaluated lor any therapeutic application, particularly in the context ol a disease or condition involving a nucleotide repeat sequence. Any candidate therapeutic agent or candidate compound may be implemented as the “agent" for achieving a particular physiological effect. In various embodiments, a candidate therapeutic agent is a small molecule, protein (peptide or antibody;, or nucleic acid. Consequently, candidate therapeutic agents may also be a small molecule, protein, or nucleic acid Such agents and compounds may be in a pharmaceutically acceptable formulation. As will be appreciated, the increases or decrease of the measured stress level (i.e. cortisol level) following administration of the candidate therapeutic agent will characterize the candidate therapeutic agent as being an agonistic or antagonistic of cortisol expression and activity in the subject.

[0206] In various embodiments, the candidate therapeutic agent may be a PTP inhibitor, PTP agonist, or an agent targeting an upstream regulator, interacting protein, or post-translational modification (e.g., oxidoreductases affecting the redox-sensitive catalytic cysteine in PTPs). The ability to monitor enzymatic activity in addition to expression level enables differentiation between agents that alter protein abundance and those that alter catalytic function, both of which may be relevant to therapeutic efficacy. In various embodiments, the candidate agent may be selected from a virtual chemical library. As used herein, the term “library" refers to a plurality of compounds A library can be a combinatorial library, e.g., a collection of compounds synthesized using combinatorial chemistry techniques, or a collection of unique chemicals of low molecular weight (less than 1000 daltons) that each occupy a unique three-dimensional space.

[0211]

[0207] In various embodiments, the method may be implemented in an in vitro system such as cultured primary cells, immortalized cell lines, or organoids derived from a subject or population of interest. In these embodiments, PTP expression or activity is measured in baseline (pre-treatment) and post-treatment samples, allowing for paired analysis and precise quantification of therapeutic impact.

[0212]

[0208] In various embodiments, the method enables structure-activity relationship (SAR) profiling during early-stage drug development. Multiple analogues of a lead compound may be tested for their capacity to modulate specific PTP isoforms using the disclosed method. Data obtained may be used to rank candidate compounds by potency, selectivity, and efficacy based on changes in PTP peptide signals or enzymatic activity readouts.

[0213]

[0209] In various embodiments, the method may be used to assess the mechanism of action of a candidate therapeutic agent. For example, a reduction in PTP enzymatic activity without a change in expression level may indicate direct inhibition of catalytic function, whereas a reduction in both activity and expression may suggest transcriptional or post-translational regulation. Conversely, increased activity following treatment with a small molecule agonist or signalling enhancer may be indicative of pathway reactivation or immune stimulation.

[0214]

[0210] Accordingly, the method provides a robust platform for drug discovery, lead optimization, and target validation in the context of diseases or conditions associated with dysregulated PTP function. The method may be further combined with downstream phenotypic assays, transcriptomic analysis, or clinical outcome data to correlate molecular changes in PTP activity with therapeutic response and safety profiles.

[0215]

[0211] The term “administering" and variations of that term including “administer" and “administration", includes contacting, applying, delivering or providing an agent, compound or composition (i.e. candidate agent) to the reporter cell by any appropriate means.

[0212] The methods disclosed herein may be an in vitro, or ex vivo method of use.

[0216]

[0213] The invention further features kits for carrying out the methods of the invention. Such kits typically comprise a number of agents and reagents useful and necessary to perform the methods of the invention.

[0217]

[0214] All embodiments disclosed herein in relation to the methods of the invention similarly apply to the kits and uses disclosed and vice versa.

[0218] EXAMPLES

[0219]

[0215] This method disclosed herein can detect and screen for protein expression and enzymatic activity of all 37 human protein tyrosine phosphatases (PTPs), from any kind of biological material, including but not exclusively cultured cell lines, organoids, and human tissue needle biopsies.

[0220]

[0216] In particular, the method involves chemical derivatization of endogenous reduced and oxidised PTPs, to capture physiological redox-regulated PTP states, followed by antibody-based pulldown of PTP catalytic sites. By targeted mass spectrometry quantification of unique catalytic site, the protein expression level of each PTP can be read out in a single measurement on 75min LC gradient. A Parallel Reaction Monitoring (PRM) targeted method holds an advantage compared to previously employed methods, allowing for the comprehensive identification of all human PTPs, in the sample.

[0221]

[0217] An adaptation of this protocol by alternative chemical derivatisation steps can allow endogenously oxidised PTPs to be identified and quantified from the same biological material. The PTP activity in the biological material can be calculated by the Total PTP subtracted by oxidised PTP.

[0222]

[0218] All steps are performed at room temperature unless stated otherwise.

[0223] Example 1 : Biological material lysis and chemical derivatization

[0224]

[0219] The cysteine of the classical cysteine based PTPs, exists in two forms within the living organisms: the active-reduced as thiolate anion state (S-) and the inactive-oxidized as sulphenic acid state (S-OH).To assess both forms of PTPs, a given sample under evaluation is divided into two pools, each serves to obtain (A) total PTPs or (B) oxidized PTPs. Two distinct lysis buffers are prepared for each pool.

[0225]

[0220] The lysis buffer for a (A) total PTPs pool contains Urea, Ammonium bicarbonate (AmbiC), Protease inhibitors, DNAse, RNAse, Sodium deoxycholate and dithiothreitol (DTT). A reducing agent dithiothreitol (DTT) is used to convert all the PTPs within the sample into a thiolate anion state (S-). Whilst the lysis buffer for a (B) oxidized PTPs pool includes the same reagents, with N-Ethylmaleimide (NEM) as a replacement of dithiothreitol (DTT). An alkylating agent N-Ethylmaleimide (NEM) is used to permanently lock all the active-reduced as thiolate anion state (S-) PTPs within the sample, leaving the inactive-oxidized as sulphenic acid (S-OH) PTPs unaltered.

[0226]

[0221] Each sample pool is mixed with the corresponding lysis buffer, sonicated and centrifuged. The supernatants are collected and reducing agent dithiothreitol (DTT) is added to the B)oxidized PTPs pool. This converts the previously unaltered inactive-oxidized as sulphenic acid state (S-OH) PTPs into thiolate anion state (S-). Both pools undergo buffer exchange, with centrifugal filters to remove Urea. Fresh hyperoxidizing agent pervanadate, which consist of H2O2 and Sodium Orthovanadate, is mixed with each pool of PTPs and left at room temperature under rotation for 15minutes in the dark. Pervanadate adds a sulfonic acid group to the previously converted (A)total PTPs and (B)oxidized PTPs pools into thiolate anion state (S-), transforming them into the permanent sulfonic acid (S-O3H) form (FIG. 2). The transformation of PTPs into a permanently inactive sulfonic acid (S-O3H) form facilitate further application of ox-PTP antibody.

[0227] Example 2: Protein digestion and peptide clean-up

[0228]

[0222] Each pool is mixed with Urea, followed by pH adjustment with NaOH. The pools then undergo 1 hour incubation with dithiothreitol (DTT), and 30-minute incubation with iodoacetamide (IAA) in the dark. Ammonium bicarbonate(AmbiC) is introduced, and digestion enzyme trypsin is added for an overnight incubation, followed by 2 more additional trypsin incubations of 4hours.. All incubations with digestion enzymes are carried out at 37°C.

[0229]

[0223] Ultra-sensitive peptide purification from each pool is carried out on reverse-phase Sep-Pak 1cc (50mg) C18 cartridges. The samples pH is adjusted with 100% formic acid and then 0.1% of formic acid is added to reach the volume of 1 ml. The C18 column is initially activated by passing through 100% acetonitrile (ACN), followed by equilibration with 0.1 % formic acid. Pools are centrifuged for 5 minutes, and the supernatants are loaded onto the columns and allowed to pass through by gravity flow. Flow through is collected and stored. Columns are washed with 0.1% formic acid and collection lo-bind tubes are placed under the columns. Finally, elution buffer containing acetonitrile (ACN) and formic acid is added to the columns, this step is repeated two times. Collected elutions are left to dry in a vacuum centrifugation and stored at -80°C.

[0230] Example 3: Antibodv-based immunoprecipitation

[0231]

[0224] Spin columns are prepared by adding an ox-PTP antibody specific for sulfonic acid (S-O3H) and A / G agarose. After 1 hour incubation with end-to-end rotation on a Ferris wheel, the columns are washed three times with PBS 1 X. Disuccinimidyl suberate (DSS) is added for 20-minute incubation, followed by three washes with PBS 1X. Purified PTP peptides are reconstituted in PBS 1 X and added into the columns with end-to-end rotation at 4°C overnight. The antibody selectively binds to PTP peptides with sulfonic acid (S-O3H). On the next day, the columns are centrifuged and the flow through is collected. 10% acetic acid is introduced to elute PTP peptides bound to the ox-PTP antibody. Afterwards, columns are washed with PBS 1X and previously collected flow through is reintroduced for an additional 3 hour incubation with subsequent elution. The elution step is repeated X4 times, and all elutions are collected in the same lo-bind tube. Collected elutions are left to dry in a vacuum centrifugation and stored at -80°.

[0232] Example 4: LC-MS / MS analysis in Parallel Reaction Monitoring (PRM) mode

[0233]

[0225] Eluted peptides are reconstituted in a solution which contains acetonitrile (ACN), trifluoroacetic acid (TFA) and acetic acid. Reconstituted components are injected into LC-MS / MS for the analysis in scheduled Parallel Reaction Monitoring (sPRM) mode. Peptide separation is carried out over a 75-minute reverse-phase gradient on Vanquish UHPLC system (Thermo). A protein separation is performed in the analytical column (PepMap RSLC C18, 2 pm, 100A, 50 cm x 75 pm) with flow rate of 300ng / ml. Gradient parameters for the solvent B (80% acetonitrile (ACN) follows as: first 60 minutes the concentration steady increase from 0 to 32%, in the next 8 minutes the concentration reach 50%, and for the final 7 minutes the concentration rise till 100% (Table 4).

[0234]

[0226] Table 4: LC settings

[0235]

[0227] Parameters present in Table 5, are used to set retention time and precursor m / z for all 37 human PTP peptides, for the MS analysis.

[0236]

[0228] Table 5: Generated list of m / z and retention time for all 37 human PTP peptides.

[0237] Example 5: Comparative Testing

[0238]

[0229] The main difference among the three methods (DDA mode, PRM mode and sPRM mode) lies in the number of detected PTP peptides and the confidence in hyperoxidation site localization. Scheduled PRM outperformed the other methods, detecting all PTP peptides with over 95% probability of correct hyperoxidation site localization. In DDA mode, 41 PTP peptides were detected with 100% hyperoxidation probability at the catalytic cysteine, except for PTPN12 (>75%) and PTPN18 (<75%), while PTPN22 was not detected. PRM mode detected 43 out of 44 peptides, but PTPN22 did not reach the hyperoxidation probability threshold (FIG. 3).

[0239]

[0230] Compared to DDA and PRM methods, scheduled-PRM significantly improved chromatographic peaks. Peaks appeared sharper within a 1 minute window, whereas in other methods peaks were not always well-defined and spanned over 2-3 minutes (FIG.4A and 5A). Additionally, transitions co-eluted more accurately within the peak (10 transition ions shown), improving peak clarity. More peptide-spectrum matches (PSMs) (shown as vertical lines FIG.4A and 5A) were detected, indicating that the spectra was assigned to a reference peptide in the spectral library, increasing accuracy and confidence in identification. The dotp values were also higher in scheduled-PRM, further improving confidence in peptide identification (FIG.4B and 5B). Ultimately, scheduled-PRM detected more synthetic peptides providing superior peptide coverage (FIG.4C and 5C).

[0240] Example 6: Cell Lines and Organoids

[0241]

[0231] Western Blotting results indicating a successful chemical derivatization and hyperoxidation of biological materials (established cell lines and liver organoid). Multiple bands can be observed for each sample. This banding pattern is expected, since the intracellular PTP repertoire spans a range of molecular weights. Fractions that represent total phosphatases (A - TOT) have more bands and stronger intensities than oxidised PTPs (B-Ox) (FIG. 6), as the oxidised PTPs are only a subset of the total PTPs.

[0232] Results from biological samples confirmed the reliability and quality of the targeted LC- MS / MS method. PTPN1 was selected as a representative example for cancer cell lines, as it was consistently detected in all lines in at least two out of three replicates (FIG. 7A-C). For the liver organoids, PTPN1 and PTPRA were selected because PTPRAwas present in both tumor and normal organoids, whereas PTPN1 was only detected in tumor organoid (FIG. 7D-I). Notably, PTPN1 was consistently detected at 33.3-34 minutes in the chromatographic gradient, while PTPRA appeared consistently at 18.6 minutes, indicating high reproducibility of its retention time, supporting robustness of the method. To confirm protein identity in the cell lines, mirror plots were generated comparing a detected PTPN1 to a synthetic PTPN1 . Matching fragment ions and similar intensities demonstrated accurate protein identification (FIG.7B,F,I). In liver organoids, mirror plots were used to compare PTPRA and PTPN1 between tumor and normal organoids. Dotp values averaged 0.89 for the PTPN1 , and 0.97 for PTPRA, confirming strong spectral similarity between experimental and reference spectra (FIG.7C,E,F). Overall, these results demonstrate that the developed method is reproducible and effective for detecting PTPs in biological systems.

[0242]

[0233] The top 3 cell lines with the highest number of detected PTPs are PC9 (21 ), SNU475 (17) and SNU1 (17) (FIG. 8A).The high expression of phosphatases in liver-derived line SNU475 aligns with expectations, given the known involvement of PTPs in MASLD and HOC. Notably, PTPN11 was consistently detected across all three cell lines (FIG. 8B). Interestingly, MS analysis revealed increased expression of PTPs in tumor tissue (13 PTPs) compared to normal tissue (3 PTPs) of liver organoids, aligning with the involvement of phosphatases in liver cancer (FIG.8A,C). Consistent with findings in the SNU475 cell line, PTPN11 was also detected in cancerous tissue of liver organoids.

[0243] Discussion

[0244] The present invention and method disclosed herein employs a scheduled parallel reaction monitoring (PRM) mode, offering a distinctive advantages in detection of all 37 human PTPs. PRM utilizes restricted mass-to-charge ratios (m / z) and retention time (RT), a pioneering feature in the context of comprehensive identification of all human PTPs.

[0245]

[0234] Compared to previously employed DDA mode, sPRM does not rely on protein abundance as it allows pre-defined selection of m / z (mass-to-charge ratio) allowing the identification of the protein of the interest, in this scenario PTPs. Moreover, sPRM offers higher identification and quantitative accuracy, due to its targeted nature. Additionally, sPRM show improvements over SRM approach. Specifically, higher specificity and identification of the peptide, as all potential transitions of a target peptide are monitored. While in SRM mode, up to 5 transitions can be monitored for each PTP, hampering accurate identification. To further increase the sensitivity and selectivity, the data acquisition in sPRM take place in a fixed retention time (RT). This feature ensures that peptides are only measured in a specific time window of chromatograph, preventing noise peaks from near - isobaric peptide ions (peptides with same m / z but different composition).

[0235] In summary, the scheduled PRM mode employed in the established technology offers superior sensitivity, selectivity compared to the DDA and PRM modes utilized previously, representing an advancement in comprehensive detection of human PTPs.

[0246]

[0236] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. Other embodiments are within the following claims.

[0247]

[0237] One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. Further, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The methods, kits and uses described herein are presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention are defined by the scope of the claims. The listing or discussion of a previously published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0248]

[0238] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, it should be understood that although the present invention has been specifically disclosed by exemplary embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0249]

[0239] The content of all documents and patent documents cited herein is incorporated by reference in their entirety.

Claims

CLAIMSWhat is claimed is:

1. A method of detecting, and optionally quantifying, the expression level and / or enzymatic activity of phosphatases (PTPs) in a sample containing, or suspected of containing, PTPs, comprising the following steps: a) processing the sample; b) isolating PTPs in the sample; and c) detecting, and optionally quantifying, the expression level and / or enzymatic activity of the PTPs in the sample using LC-MS / MS analysis in scheduled PRM mode.

2. The method of claim 1 , wherein the expression level and / or enzymatic activity of the PTPs identified, and optionally quantified, in the sample, is compared to the expression level and / or enzymatic activity of the PTPs identified, and optionally quantified, in a control or comparative sample.

3. The method of claim 1 or 2, wherein step a) comprises converting PTPs in the sample into an inactive sulfonic acid state (S-OsH).

4. The method of any one of claims 1 -3, wherein step a) comprises chemical derivatization of the sample with a reducing agent and / or an alkylating agent.

5. The method of claim 4, wherein the chemical derivatization converts both total PTPs and oxidized PTPs within the sample into S- state, with subsequent hyperoxidation carried out to convert the S- state PTPs into an inactive sulfonic acid state (S-OsH).

6. The method of any one of claims 1 -5, wherein step a) comprises dividing the sample into two pools of sample pool (A) for obtaining total PTPs and sample pool (B) for obtaining oxidized PTPs.

7. The method of claim 6, wherein sample pool (A) and (B) are treated with lysis buffers, preferably the lysis buffer used for sample pool (A) is distinct from the lysis buffer used for sample pool (B), more preferably the lysis buffer used for sample pool (A) comprises dithiothreitol (DTT) and the lysis buffer used for sample pool (B) comprises N-Ethylmaleimide (NEM).

8. The method of claim 7, wherein following treatment with the lysis buffers, sample pool (A) and (B) are hyperoxidated, preferably pervanadate is mixed with each sample pool (A) and (B) under suitable conditions to convert all PTPs into a permanently inactive sulfonic acid state (S-OaH).

9. The method of any one of claims 1 -8, wherein the isolation of the PTPs comprises antibodybased immunoprecipitation, preferably the antibody is specific for PTPs in an oxidized state,preferably the antibody is an ox-PTP antibody and the PTPs are in an inactive sulfonic acid state (S- OaH), and the ox-PTP antibody is specific for sulfonic acid (S-OaH), more preferably the immunoprecipitation comprises crosslinking of the ox-PTP antibody.

10. The method of any one of claims 1 -9, wherein step a) further comprises a step of protein digestion and peptide purification of the sample.

11. The method of any one of claims 1 -10, wherein step c) comprises detecting, and optionally quantifying, the enzymatic activity of PTPs in the sample, and subtracting the oxidized PTP from total PTP, wherein the oxidized PTPs indicate the number of inactive PTPs in the sample with the remaining PTPs in the total PTPs indicating the number of active PTPs in the sample.

12. The method of any one of claims 1 -11 , wherein the LC-MS / MS analysis comprises a reverse- phased chromatography, preferably using a UHPLC system, more preferably the LC comprises a 75 minute gradient.

13. The method of any one of claims 1 -12, wherein step c) comprises detecting, and optionally quantifying, one or more PTP peptides in the sample, preferably the one or more PTP peptides comprise a PTP catalytic site, wherein the PTP catalytic site comprises a hyperoxidized cysteine residue in the PTP catalytic site.

14. The method of any one of claims 1 -13, wherein the scheduled PRM mode uses pre-defined and selected mass-to-charge ratios (m / z) and retention time (RT) windows for the PTPs, preferably for PTP peptides.

15. The method of claim 14, wherein the retention time is configured to prevent noise peaks from near - isobaric peptide ions, and to increase the number of transitions that can be obtained for each peptide, enhancing quantification.

16. The method of claim 14, wherein mass-to-charge ratios for +2, +3, and +4 charge states of the PTP peptides are used for the scheduled PRM.

17. The method of any one of claims 1 -16, wherein step c) further comprises distinguishing between each PTP identified in the sample.

18. The method of any one of claims 1 -17, wherein a precursor ion spectrum and / or a fragment ion spectrum is obtained from the LC-MS / MS run to detect, and optionally quantify, the expression level and / or enzymatic activity of PTPs in the sample.

19. The method of any one of claims 1 -18, wherein the PTPs are human PTPs, and the method is capable of detecting and distinguishing between all 37 human PTPs.

20. The method of any one of claims 1 -19, wherein parameters of the LC-MS / MS analysis in scheduled PRM mode are configured to detect and distinguish between human PTPs based on a PTP amino acid sequence upstream of a PTP catalytic site comprised in PTP peptides, preferably the parameters include:(i) selected PTP peptide sequences corresponding to each human PTP, preferably the PTP peptide comprise the PTP amino acid sequence upstream of the PTP catalytic site;(ii) modification of the PTP peptide sequence (i), preferably modify a cysteine (C) residue in the PTP catalytic site to add 48 Daltons (Da) to the molecular weight of the peptide (i));(iii) charge state (z) for each PTP peptide sequence (i);(iv) precursor m / z of each PTP peptide sequence (i);(v) retention time of each PTP peptide sequence (i);(vi) adjusted retention time; and / or(vii) minimum start time and minimum end time related to the RT.

21. The method of claim 20, wherein the parameters of the LC-MS / MS analysis in scheduled PRM mode of the human PTPs, are defined in Table 5, such that the method is capable of detecting and distinguishing between 37 human PTPs in a sample.

22. The method of any one of claims 1 -21 , wherein the sample is a biological sample that has been obtained from a subject, and the expression level and / or enzymatic activity of PTPs identified, and optionally quantified, in the biological sample is compared with reference expression levels and / or enzymatic activity of PTPs associated with a disease or condition associated with one or more PTPs.

23. The method of claim 22, wherein each reference expression levels and / or enzymatic activity is specific for a disease or condition associated with one or more PTPs.

24. A method for the diagnosis, prognosis, or monitoring of a disease or condition associated with one or more PTPs in a subject, comprising detecting, and optionally quantifying, the expression level and / or enzymatic activity of protein tyrosine phosphatases (PTPs) in a biological sample that has been obtained from the subject, using the method according to any one of claims 1 -23, wherein the detected, and optionally quantified, expression level and / or enzymatic activity of protein tyrosine phosphatases (PTPs) assists in the diagnosis, prognosis, or monitoring of a disease or condition associated with one or more PTPs.

25. A method for assisting in determining a subject's risk for developing a disease or condition associated with one or more PTPs, comprisingdetecting, and optionally quantifying, the expression level and / or enzymatic activity of protein tyrosine phosphatases (PTPs) in a biological sample that has been obtained from the subject, using the method according to any one of claims 1 -23, wherein the detected, and optionally quantified, expression level and / or enzymatic activity of protein tyrosine phosphatases (PTPs) is indicative of the subject's risk for developing a disease or condition associated with one or more PTPs.

26. A method for predicting a subject' s response to a therapeutic treatment for treating a disease or condition associated with one or more PTPs, comprising detecting, and optionally quantifying, the expression level and / or enzymatic activity of protein tyrosine phosphatases (PTPs) in a biological sample that has been obtained from the subject, using the method according to any one of claims 1 -23, wherein the detected, and optionally quantified, expression level and / or enzymatic activity of protein tyrosine phosphatases (PTPs) is predictive of the subject's response to the therapeutic treatment.

27. A method for monitoring treatment efficacy in a subject undergoing treatment for a disease or condition associated with one or more PTPs comprising, detecting, and optionally quantifying, the expression level and / or enzymatic activity of protein tyrosine phosphatases (PTPs) in a biological sample that has been obtained from the subject at different time points before, during, or after treatment, using the method according to any one of claims 1 -23, optionally, the detecting step is repeated two or more times before, during, or after treatment within a time-frame and the detection at each time point is compared against each other to assess the progression of the disease or condition and efficacy of the treatment.

28. The method of any one of claims 23-27, wherein the disease or condition associated with one or more PTPs are known to be associated with aberrant expression and / or activity level of one or more PTPs, and comprises cancer, a metabolic disorder, an autoimmune disease, a neurological disorder, an infectious disease, a cardiovascular disease, a developmental disorder, and an inflammatory disease.

29. A method for developing or identifying therapeutic agents capable of modifying the expression and / or activity level of PTPs in a subject, the method comprising: a) measuring the expression level and / or enzymatic activity of protein tyrosine phosphatases (PTPs) in a first biological sample obtained from the subject, with the method of any one of claims 1 - 23, b) administering a candidate therapeutic agent to the subject,c) measuring the expression level and / or enzymatic activity of protein tyrosine phosphatases (PTPs) in a second biological sample obtained from the subject after step b), with the method of any one of claims 1 -23, following the administration of the candidate agent, d) characterizing the candidate therapeutic agent as capable of modifying the activity of PTPs, if the subject's measured expression level and / or enzymatic activity of PTPs increases or decreases following administration of the candidate therapeutic agent.

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