Peptide quantitation assay that distinguishes full-length high molecular weight kininogen (HMWK) from cleaved HMWK

Abstract

Provided herein are methods for distinguishing full-length high molecular weight kininogen (HMWK) and cleaved HMWK in a sample. Such methods can comprise treating a biological sample with a protease to produce a plurality of digested peptides and measuring one or more tag peptides indicative of cleaved HMWK and / or full-length HMWK.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 267,743, filed December 15, 2015, the entire contents of which are incorporated herein by reference. Background Technology

[0003] Kininogen is a precursor to kinins such as bradykinin and kallikrein. Two types of human kininogen exist: high molecular weight kininogen (HMWK) and low molecular weight kininogen (LMWK), which are splice variants. HMWK primarily functions as a cofactor in coagulation and inflammation and is a preferred substrate for plasma kallikrein-mediated bradykinin production. Both HMWK and LMWK are cysteine ​​protease inhibitors.

[0004] HMWK is a circulating plasma protein involved in blood clotting. HMWK can also generate vasodilatory bradykinin through the bradykinin system. HMWK adheres to cell surface receptors on endothelial cells, monocytes, and platelets, leading to localized coagulation and bradykinin production. The active peptide bradykinin released from HMWK exhibits various physiological effects, such as smooth muscle contraction, hypotension, polyuria, and decreased blood glucose levels. It is a regulator of inflammation and has cardioprotective effects, acting directly through bradykinin and indirectly through vasodilators derived from endothelial cells. Bradykinin is a major regulator of pain, inflammation, edema, and angiogenesis.

[0005] Plasma kallikrein (pKal) is the major bradykinin-producing enzyme in circulation. Activation of pKal occurs through a contact system associated with a disease pathology related to hereditary angioedema (HAE). pKal cleaves HMWK (a single-chain polypeptide) to produce bradykinin and a cleaved form of HMWK containing two polypeptide chains (heavy and light chains) held together by disulfide bonds. (Cugno et al., Blood (1997) 89: 3213-3218). The light chain in the initially cleaved HMWK is approximately 56 kDa, and it can be further cleaved to form a shorter form of 46 kDa.

[0006] During an episode of hereditary angioedema (HAE), cleaved HMWK increases to approximately 47% of total kininogen, making it a biomarker for monitoring HAE episodes (Cugno et al., 1997). Therefore, it is meaningful to develop a sensitive and reliable assay for detecting the level of cleaved HMWK in biological samples. Summary of the Invention

[0007] This disclosure is based, at least in part, on the development of a sensitive and selective assay method, which may involve liquid chromatography-mass spectrometry (LC-MS), for example, using multiple reaction monitoring (MRM) to distinguish between full-length HMWK and cleaved HMWK. Such an assay method utilizes a tag peptide indicating cleaved HMWK (e.g., a C-terminal peptide from the heavy chain or an N-terminal peptide from the light chain) and / or full-length HMWK (e.g., relative to low molecular weight kininogen).

[0008] Therefore, one aspect of this disclosure provides a method for detecting cleaved high molecular weight kininogen (HMWK) in a sample, the method comprising: (i) providing a sample suspected of containing HMWK; (ii) contacting the sample with a protease to generate a plurality of digestible peptides; and (iii) measuring the level of a tagged peptide in the plurality of digestible peptides, wherein the tagged peptide indicates cleaved HMWK.

[0009] In some embodiments, the tagged peptide indicates the 46 kDa light chain of cleaved HMWK, including but not limited to: KHNLGHGH (SEQ ID NO:1), KHNLGHGHKHE (SEQ ID NO:2), KHNLGHGHK (SEQ ID NO:3), or KHNLGHGHKHER (SEQ ID NO:4).

[0010] In some implementations, the tagged peptide indicates the 56kDa light chain of cleaved HMWK, for example, SSRIGE (SEQ ID NO:5).

[0011] The method described herein may further include measuring the level of a tagged peptide indicating full-length HMWK, including but not limited to: GHEKQRKH (SEQ ID NO:6), KQRKHNLGHGHKHE (SEQ ID NO:7), DWGHKQRKHNLGHGHKHER (SEQ ID NO:8), HNLGHGHK (SEQ ID NO:9), or SYYFDLTDGLS (SEQ ID NO:10).

[0012] In another aspect, this disclosure is characterized by a method for distinguishing between full-length high molecular weight kininogen (HMWK) and cleaved HMWK in a sample. The method includes: (i) providing a sample suspected of containing full-length HMWK and / or cleaved HMWK; (ii) contacting the sample with a protease to generate a variety of digestible peptides; (iii) measuring the level of a first digestible peptide (e.g., SSRIGE: SEQ ID NO: 5) obtained from step (ii), wherein the first digestible peptide is specific to cleaved HMWK (e.g., a tagged peptide of cleaved HMWK) compared to full-length HMWK; (iv) measuring the level of a second digestible peptide obtained from step (ii), wherein the second digestible peptide (e.g., SYYFDLTDGLS: SEQ ID NO: 10) is specific to HMWK (e.g., a tagged peptide of HMWK) compared to low molecular weight kininogen (LMWK); (v) determining a ratio between the first digestible peptide and the second digestible peptide; and (vi) distinguishing between cleaved HMWK and full-length HMWK in the sample based on the ratio determined in step (v).

[0013] In any of the assay methods described herein, the protease used to digest HMWK in the sample can be an intracellular protease such as chymotrypsin, Glu-C, Asp-N, cathepsin G, or Lys-C. In some embodiments, any tagged peptide can be measured by liquid chromatography-mass spectrometry (LC-MS), such as MRM-MS.

[0014] The sample to be analyzed by any of the assays described herein is a biological sample (e.g., blood, plasma, or serum sample) obtained from a human subject, such as a human patient with or suspected of having hereditary angioedema (HAE). In some instances, the biological sample may be a normal plasma sample, an FXIIa-activated plasma sample, or an unactivated plasma sample. When the biological sample is a plasma sample, it may be collected in a vacuum-sealed blood collection tube (e.g., a sample collection anticoagulant tube, “SCAT tube”) comprising a liquid formulation containing a mixture of protease inhibitors.

[0015] In some embodiments, the protease digestion step of any of the assays described herein can be performed in the presence of a reducing agent, such as DTT, BME, or TCEP. For example, the biological sample can be incubated with the reducing agent at 90°C for 1 hour. In some instances, the protease digestion step can be performed in the absence of a protease inhibitor, an anticoagulant (e.g., citrate), or both a protease inhibitor and an anticoagulant. In some instances, the protease / protein ratio is approximately 1:20, for example, Glu C / protein is 1:20.

[0016] Any of the described assay methods can be applied to the diagnosis and / or prognosis of HAE. Accordingly, this disclosure also provides methods for identifying human subjects with HAE or human subjects at risk of HAE attacks. In some embodiments, whether a human subject has HAE or is at risk of an HAE attack can be determined by the level of cleaved HMWK (e.g., heavy-light chain dimer, heavy chain or light chain, such as 46kDa light chain or 56kDa light chain) as determined by the assay methods described herein, wherein elevated levels of cleaved HMWK indicate HAE or a risk of an HAE attack. In other embodiments, whether a human subject has HAE or is at risk of an HAE attack can be determined by the ratio between the tagged peptide of cleaved HMWK and the tagged peptide of HMWK as determined by the assay methods described herein, wherein elevated levels of such a ratio indicate HAE or a HAE attack.

[0017] Details of one or more embodiments of the invention are set forth in the following description. Other features or advantages of the invention will become apparent from the following drawings and detailed descriptions of several embodiments, as well as the appended claims. Attached Figure Description

[0018] Figure 1 A graph illustrating exemplary peptides (e.g., 56 kDa light chains) obtained by enzymatic digestion of cleaved HMWK found in plasma and commercially available HMWK(HK) samples by MRM analysis at optimized temperatures. SEQ ID NOs: 5, 19, 24, and 21 are depicted repeatedly from top to bottom in this graph.

[0019] Figure 2 A graph illustrating the peptides produced by digestion of HMWK standards with Glu C at 25°C and 37°C, obtained from Sigma (HK_Sigma) or Enzyme Research (HK_Enzyme Research), is shown. The graph depicts SEQ ID NOs: 5, 19, 24, 30, 29, and 7 from top to bottom.

[0020] Figure 3 Includes a graph showing the quantification of the 56 kDa light chain peptide SSRIGE (SEQ ID NO:5) in plasma samples derived from normally digested protease-digested and HAE plasma samples using targeted assays of MRM.

[0021] Figure 4 This includes a graph showing the quantification of HMWK long peptide in normal plasma samples and HAE plasma samples using targeted assays of MRM.

[0022] Figure 5A graph showing the ratio of SSRIGE (SEQ ID NO:5) / SYYFDLTDGLS (SEQ ID NO:10) in Glu C-digested HMWK and plasma samples.

[0023] Figure 6 A graph showing the average ratio of SSRIGE (SEQ ID NO:5) / SYYFDLTDGLS (SEQ ID NO:10) using the area under the peak of the curve.

[0024] Figure 7 A graph showing the concentration of HMWK (expressed as a 46 kDa light chain) cleaved in HAE patients and healthy individuals is presented. Detailed Implementation Plan

[0025] Plasma kallikrein (PKal) is a serine protease component of the contact system and is the major bradykinin-producing enzyme in circulation. The contact system is activated upon exposure to exogenous or negatively charged surfaces via factor XIIa (the active form of factor XII or FXII) or on endothelial cell surfaces via prolyl carboxypeptidase (Sainz IM et al., ThrombHaemost 98, 77-83, 2007). Activation of PKal amplifies intrinsic coagulation through feedback activation of its factor XII, and proteolytically cleaves kininogen precursors, high molecular weight kininogen (HMWK), releasing the pro-inflammatory nonapeptide bradykinin and cleaved HMWK, which contains two polypeptide chains linked by disulfide bonds (also known as 2-chain HMWK).

[0026] As the primary kininogen in circulation, plasma kallikrein is mainly responsible for the generation of bradykinin in the vascular system. A genetic defect in C1-inhibitor protein (C1-INH) results in hereditary angioedema (HAE). Patients with HAE suffer acute attacks of painful edema, often triggered by unknown factors (Zuraw BL et al., N Engl J Med 359, 1027-1036, 2008). The plasma kallikrein-kinin system (plasma KKS) has been investigated in a variety of diseases using pharmacological agents or genetic studies in animal models.

[0027] High molecular weight kininogen (HMWK) exists in plasma as a single polypeptide (1-chain) multi-domain (domains 1-6) protein with a molecular weight of approximately 110 kDa. HMWK is cleaved by pKal within domain 4 to release a 9-amino acid pro-inflammatory peptide—bradykinin—and the 2-chain form of HMWK (cleaved kininogen). The two chains of HMWK consist of a heavy chain containing domains 1-3 and a light chain containing domains 5 and 6. The heavy chain has a molecular weight of approximately 65 kDa, while the light chains exist in two forms with molecular weights of approximately 56 kDa and 46 kDa.

[0028] Levels of cleaved HMWK (e.g., 2-chain HMWK) have been found to be elevated in HAE episodes and other pKal-related diseases. Therefore, cleaved HMWK could serve as a biomarker for monitoring disease progression and / or therapeutic efficacy. However, suitable reagents and / or appropriate assays are lacking in the art to effectively distinguish between intact HMWK and its cleaved forms.

[0029] This disclosure is based, at least in part, on the discovery of tag peptides indicating cleaved HMWKs, such as tagged peptides of the light chain (e.g., a 46 kDa light chain) of 2-chain HMWKs and tag peptides indicating HMWKs (e.g., full-length HMWKs). Unless otherwise specified, the term "cleaved HMWK" refers to the 2-chain dimer, the heavy chain of the dimer, or the light chain (including 56 kDa and 46 kDa light chains) described herein. Based on the discovery of such tag peptides, sensitive and selective assay methods have been developed to measure cleaved HMWKs relative to HMWKs. The assay methods described herein can be used for clinical applications (e.g., diagnosis or prognosis of HAEs) and non-clinical applications, such as for research and preclinical drug development purposes.

[0030] I. Methods for measuring HMWK pyrolysis

[0031] In some respects, this paper describes methods for measuring cleaved HMWK in a sample, for example, distinguishing between cleaved HMWK and full-length HMWK in a sample. This method may involve treating a suitable sample suspected of containing full-length HMWK, cleaved HMWK, or both full-length and cleaved HMWK with an appropriate protease to produce a variety of digestible peptides, and measuring the levels of one or more tagged peptides indicating cleaved HMWK and / or full-length HMWK. The level of cleaved HMWK, or the ratio between cleaved and full-length HMWK, can be used to indicate the activity of pKal in the sample, which is associated with HAE or the risk of HAE onset.

[0032] (i) Full-length HMWK and fragmented HMWK

[0033] The human gene encoding HMWK is kininogen 1 (KNG1). KNG1 is transcribed and optionally spliced ​​to form mRNA encoding HMWK or low molecular weight kininogen (LMWK). Exemplary protein sequences of HMWK and LMWK are provided below (the bradykinin region is highlighted and bolded):

[0034] >gi|156231037|ref|NP_001095886.1|Kallikrein-1 isotype 1 precursor [Homo sapiens]

[0035]

[0036] >gi|4504893|ref|NP_000884|Kallikrein-1 isoform 2 precursor [Homo sapiens]

[0037]

[0038] The following are exemplary sequences of the heavy and light chains of a cleaved kininogen:

[0039] >Cleavage of the kininogen-1 heavy chain (the italicized region in SEQ ID NO:11 above)

[0040]

[0041] >Cleavage of kininogen-1 light chain (56 kDa) (in bold in SEQ ID NO:11 above)

[0042]

[0043]

[0044] >Cleavage of kininogen-1 light chain (46KD) (italicized and bolded in SEQ ID NO:11 above)

[0045]

[0046] (ii) Sample preparation

[0047] Any sample that may contain HMWK (e.g., full-length HMWK, lysed HMWK, or both) can be analyzed using the methods described herein. As used herein, "sample" refers to a composition that may contain the analyte of interest (HMWK in this application). Samples may contain tissue from a subject, such as blood, plasma, or protein. Samples may include initial unprocessed samples taken from a subject as well as subsequently processed samples, such as partially purified or preserved forms, for example, by immunoprecipitation. Exemplary samples include blood, plasma, tears, or mucus. In other instances, samples may be compositions for in vitro analysis.

[0048] In some embodiments, the sample is a bodily fluid sample, such as a serum sample or a plasma sample. Such a sample may be a biological sample obtained from the subject to be analyzed. The terms "patient," "subject," or "host" (which may be used interchangeably) to be treated by the methods of this disclosure may refer to a human or a non-human animal. In some examples, the subject is a human patient who may have a disease associated with the exposure system, is suspected of having a disease associated with the exposure system, or is at risk of having a disease associated with the exposure system. For example, a human patient may have a prior history of HAE or may be at risk of HAE. Such a human patient may be pre-treated or undergoing drug treatment that targets components of the exposure system (e.g., pKal or FXIIa or high molecular weight kininogen).

[0049] Biological samples can be bodily fluid samples, such as blood or plasma samples. Plasma samples used in the methods described herein can be collected and processed in vacuum-sealed blood collection tubes (e.g., sample collection anticoagulant tubes or SCAT tubes), which are commonly used in medical practice to collect blood samples for various purposes. The tubes described herein can be non-glass tubes comprising liquid formulations containing a protease inhibitor cocktail.

[0050] In some embodiments, the protease inhibitor mixture may comprise at least one serine protease inhibitor and at least one cysteine ​​protease inhibitor. The at least one serine protease inhibitor may be a plasma kallikrein inhibitor. Such a protease inhibitor mixture may comprise multiple (e.g., 2, 3, 4, or 5) serine protease inhibitors, at least one of which may be a trypsin inhibitor or a human plasmin inhibitor. Preferably, the protease inhibitor mixture described herein is substantially free of protease inhibitors unstable in aqueous solution, i.e., the activity of protease inhibitors unstable in aqueous solution is non-substantial relative to the overall inhibitory activity of the protease mixture. In some examples, the amount of protease inhibitors unstable in aqueous solution may be less than 5% (w / w) of the total protease inhibitors in the mixture, for example, less than 2%, less than 1%, or less than 0.5%. In some examples, the protease inhibitor mixture is completely free of protease inhibitors unstable in aqueous solution (e.g., aqueous solution at pH 4-6). An example of a protease inhibitor unstable in aqueous solution is PPACK II, also known as HD-Phe-Phe-Arg-chloromethyl ketone.

[0051] The table below lists exemplary serine protease inhibitors, cysteine ​​protease inhibitors, and trypsin inhibitors that can be used to prepare the protease inhibitor mixtures described herein.

[0052]

[0053]

[0054]

[0055] In some instances, the protease inhibitor mixture contained in a vacuum-sealed blood collection tube comprises at least one (e.g., 1, 2, or 3) serine protease inhibitors, which may include at least one (e.g., 1, 2, or 3) trypsin / fibrinolytic enzyme inhibitors, or at least one (e.g., 1, 2, or 3) cysteine ​​protease inhibitors. Such a protease inhibitor mixture may comprise three serine protease inhibitors (e.g., benzalkonium chloride, AEBSF, and trypsin / fibrinolytic enzyme inhibitors such as soybean trypsin inhibitors) and one cysteine ​​protease inhibitor (e.g., leucopeptide).

[0056] In other examples, the protease inhibitor mixture may contain at least one serine protease inhibitor (e.g., a plasma kallikrein inhibitor) and at least one cysteine ​​protease inhibitor (e.g., leucopeptide). The plasma kallikrein inhibitor may be EPI-KAL2 (Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp GlyPro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys GluGlu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu GluCys Lys Lys Met Cys Thr Arg Asp; SEQ ID NO:16), a specific plasma kallikrein inhibitor, a recombinant protease inhibitor that allows the tube to contain reagents that permit the assay of activated plasma kallikrein using methods such as immunoassays.

[0057] Any mixture of protease inhibitors can be dissolved in a suitable solution to form a liquid formulation. Such a suitable solution can be an acid-citrate-dextrose solution containing trisodium citrate, citric acid, and dextrose. The solution can have a pH of approximately 4-6, for example, pH 4.5. The liquid formulation may further contain cationic polymers such as hymexazolium bromide molecules. It reduces contact system activation by interacting with negatively charged surfaces; and chelating agents (e.g., EDTA) can inhibit metalloproteinases.

[0058] The concentration of each protease inhibitor in the mixture can be 5x or 10x the final concentration at which the inhibitor is used to inhibit the corresponding protease, depending on the dilution factor in practice. The final concentrations of specific commercial protease inhibitors are known in the art and can be obtained according to the manufacturer's formulation. In some instances, the concentration range of EPI-KAL2 can be 5 μM–15 μM (e.g., 5 μM–10 μM or 10 μM–15 μM), the concentration range of leucopeptide can be 200 μM–300 μM (e.g., 200 μM–250 μM, 240 μM–270 μM or 250 μM–300 μM); the concentration range of soybean trypsin inhibitor can be 1 mg / ml–3 mg / ml (e.g., 1 mg / ml–2 mg / ml or 2 mg / ml–3 mg / ml); the concentration range of benzalkonium chloride can be 80 mM–120 mM (e.g., 80 mM–100 mM or 100 mM–120 mM); and / or the concentration range of AEBSF can be 10 mM–30 mM (e.g., 10 mM–20 mM or 20 mM–30 mM).

[0059] When using peptide-based protease inhibitors (e.g., EPI-KAL2), the inhibitor can be biotinylated according to conventional methodologies. For example, the peptide inhibitor can be biotinylated as follows: Briefly, the peptide inhibitor is dissolved in a suitable solution such as phosphate-buffered saline (PBS). Freshly prepared sulfonyl-NHS-LC-biotin can be added to the peptide inhibitor solution and incubated on ice for an appropriate time. Excess unreacted and hydrolyzed biotin can be removed using a rotary desalting column. The labeling of the peptide inhibitor can be confirmed by ELISA, and the protein concentration can be determined using the Bradford assay.

[0060] Any of the liquid formulations described herein can be prepared by conventional methods, such as dissolving the appropriate components in a suitable solution and placing them in a vacuum-sealed blood collection tube, preferably a non-glass tube. The collection tube can be stored at -20°C and thawed on ice or in a refrigerator for an appropriate period before use.

[0061] In specific instances, the vacuum-sealed blood collection tubes used in the methods described herein are SCAT tubes, including SCAT 169 and SCAT 153, detailed below:

[0062] ●SCAT169: A 5mL vacuum-sealed plastic tube containing (0.5mL): 100mM benzidine, 400μg / mL, dissolved in acid-citric acid-dextrose (100mM trisodium citrate, 67mM citric acid, and 2% dextrose, pH 4.5). 2 mg / mL soybean trypsin inhibitor, 20 mM EDTA, 263 μM leucopeptide, and 20 mM MAEBSF (4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride).

[0063] ●SCAT153: A 5mL vacuum-sealed plastic tube containing (0.5mL): 10μM biotinylated EPI-KAL2, 400μg / mL, dissolved in acid-citric acid-dextrose (100mM trisodium citrate, 67mM citric acid, and 2% dextrose, pH 4.5). 20mM EDTA and 263μM leucinogen inhibitory peptide.

[0064] Following blood collection, the blood sample is processed within an appropriate timeframe (e.g., no more than 1 hour) to prepare a plasma sample. The plasma sample can be further analyzed to assess characteristics related to the subject's contact system from which the initial blood sample was obtained.

[0065] (iii) Protease digestion

[0066] Biological samples described herein, such as whole plasma samples prepared according to the methods described herein, can be treated with appropriate proteases to produce a variety of digestible peptides. Alternatively, HMWK proteins, including full-length and cleaved forms, can be enriched from the sample prior to protease digestion, for example by an immunoprecipitation reaction, or by methanol crash denaturation.

[0067] Any suitable protease can be used in the methods described herein. Preferably, the protease cleaves the protein at a specific motif / residue, such that the digestible peptide can be identified based on the protein's amino acid sequence. In some instances, the protease used in this method cleaves after a glutamate residue. Exemplary proteases include, but are not limited to, the intracellular protease Glu-C or cathepsin G. In other instances, the protease used in the assay methods described herein cleaves before an aspartic acid residue, such as the endonuclease Asp-N; or after a lysine residue, such as the endonuclease Lys-C. In yet another instance, the protease used in the assay methods described herein cleaves after an amino acid residue carrying a large hydrophobic side chain (e.g., tyrosine, tryptophan, or phenylalanine). An example of such a protease is chymotrypsin.

[0068] The protease lysis reaction can be performed under appropriate conditions that allow for the complete digestion of full-length and cleaved HMWK proteins in the sample. In some embodiments, the protease lysis reaction mixture may contain a suitable concentration (e.g., 1 mM, 0.5 mM, 0.1 mM, or 0.05 mM) of a reducing agent, such as dithiothreitol (DTT), β-mercaptoethanol (BME), or tris(carboxyethyl)phosphine (TCEP). For example, when using DTT, its concentration may be 0.1 mM or 0.05 mM. The protease lysis reaction can be carried out at an appropriate temperature (e.g., 25°C, 37°C, 50°C, 55°C, or 90°C) for an appropriate time period (e.g., 30 min, 60 min, -90 min, or 180 min).

[0069] In some instances, the mixture containing the reducing agent may be incubated at a high temperature for an appropriate period (e.g., 30 or 60 minutes at 55°C; or 30 or 60 minutes at 90°C) to allow complete denaturation of the proteins in the sample. A protease may then be added to the mixture, and the digestion reaction may be performed at an appropriate temperature for an appropriate period. The precise digestion temperature / time will depend on the specific protease used in this method, which is within the knowledge of those skilled in the art. In one instance, Glu C enzyme is used, and the digestion reaction may be carried out overnight at 25°C or 37°C (e.g., 12, 14, 17, or 19 hours). In another instance, chymotrypsin is used, and the digestion reaction may be carried out at 40°C–60°C (e.g., 50°C) for an appropriate period, such as 2–5 hours (e.g., 3 hours).

[0070] The reaction mixture may be free of protease inhibitors, anticoagulants (such as citrate), or both. The enzyme / protein ratio in the reaction can range from 1:5 to 1:30, for example, 1:5, 1:10, 1:20, 1:25, or 1:30. Specific reaction conditions, including temperature, reaction time, enzyme / protein ratio, presence / absence of protease inhibitors, presence / absence of anticoagulants, and presence / absence of reducing agents, can depend on the type of protease used and the sample to be treated, and can be determined by methods known in the art or those described herein.

[0071] (vi) Measurement of tagged peptides

[0072] A tagged peptide is a digestible peptide produced from a protease reaction that is specific to a particular type of kininogen compared to other types of kininsogens. Such peptides can be identified based on the specificity of the protease used in the methods described herein and the amino acid sequences of the different types of kininsogens. See, as disclosed herein. A peptide (tagged peptide) specific to a first type of kininogen (such as a full-length kininogen) relative to a second type of kininogen (such as a cleaved kininogen) is a peptide that can only be produced by cleaving a first type of kininogen using a protease and cannot be produced by cleaving a second type of kininogen using the same protease. For example, a tagged peptide of cleaved HMWK (e.g., the heavy or light chain of cleaved HMWK, such as a 46 kDa light chain) is a peptide that can only be produced by digesting cleaved HMWK (e.g., the heavy or light chain of cleaved HMWK) with a protease and cannot be produced by digesting full-length HMWK with the same protease. Similarly, a tagged peptide of full-length HMWK is a peptide that can only be produced by digesting single-chain HMWK with a protease and cannot be produced by digesting cleaved HMWK with the same protease. HMWK-tagged peptides refer to peptides produced by digesting HMWK (single-chain HMWK, double-chain HMWK, or both) with a protease, but not by digesting LMWK with the same protease.

[0073] Exemplary tagged peptides for cleavage of HMWK include: (a) 56 kDa light chain tagged peptides, such as SSRIGE (SEQ ID NO:5), which can be produced by Glu-C digestion, and (b) 46 kDa light chain tagged peptides, such as KHNLGHGH (SEQ ID NO:1), KHNLGHGHKHE (SEQ ID NO:2), KHNLGHGHKHER (SEQ ID NO:4), or KHNLGHGHK (SEQ ID NO:3), which can be produced by chymotrypsin digestion, Glu-C digestion, Asp-N digestion, and Lys-C digestion, respectively.

[0074] Table 1 below lists exemplary tag peptides of the 46 kDa light chain of HMWK for cleavage, produced by different proteases:

[0075] Table 1: Tag peptides derived from HMWK digested with multiple enzymes:

[0076]

[0077]

[0078] Exemplary tagged peptides of HMWK (such as full-length HMWK) include, but are not limited to: GHEKQRKH (SEQ ID NO:6), which can be produced by chymotrypsin digestion; KQRKHNLGHGHKHE (SEQ ID NO:7) and SYYFDLTDGLS (SEQ ID NO:10), which can be produced by Glu-C digestion; DWGHKQRKHNLGHGHKHER (SEQ ID NO:8), which can be produced by Asp-N digestion; and HNLGHGHK (SEQ ID NO:9), which can be produced by Lys-C digestion.

[0079] The level of the digestible peptide of interest can be measured using appropriate methods as known in the art or described herein. In some embodiments, the peptide of interest can be determined by immunoassay using antibodies specific to the peptide of interest, such as antibodies specific to KHNLGHGH (SEQ ID NO:1), SSRIGE (SEQ ID NO:5), and / or SYYFDLTDGLS (SEQ ID NO:10). Immunoassays that can be used to assess the level of the peptide of interest described herein include Western blotting, enzyme-linked immunosorbent assay (ELISA) (e.g., sandwich ELISA), radioimmunoassay, electrochemiluminescence-based assays, and related techniques. For example, Western blotting assays may further involve the use of quantitative imaging systems, such as commercially available LICOR imaging technology (see, e.g., from LI-COR Biosciences). (CLx infrared imaging system). In some embodiments, electrochemiluminescence detection methods or methods relying on a combination of electrochemiluminescence and patterned array techniques are used (e.g., ECL or MULTI-ARRAY techniques from Meso Scale Discovery (MSD)).

[0080] As used herein, the terms “measuring” or “measurement” or alternatively “detecting” or “detection” mean assessing the presence, absence, quantity, or amount (which may be an effective amount) of a substance in a sample, including deriving a qualitative or quantitative concentration level of such a substance, or otherwise assessing the value or classification of a subject.

[0081] "Specific binding" of an antibody to a peptide of interest is a well-known term in the art, and methods for determining such specific binding are also well-known in the art. An antibody is considered to exhibit "specific binding" if it reacts or associates with a particular target peptide more frequently, more rapidly, for a longer duration, and / or with a higher affinity than it does with an alternative peptide. It can also be understood from reading this definition that, for example, an antibody that specifically binds to a first target peptide may specifically or preferentially bind to a second target peptide, or may not specifically or preferentially bind to a second target peptide. Therefore, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, mentioning binding implies preferential binding. In some instances, an antibody that "specifically binds" to a target peptide or its epitope may not bind to other peptides or other epitopes in the same antigen.

[0082] As used herein, the term "antibody" refers to a protein that includes at least one immunoglobulin variable domain or an immunoglobulin variable domain sequence. For example, an antibody may include a heavy (H) chain variable region (abbreviated herein as V). H ) and light (L) chain variable region (abbreviated as V in this article) L In another example, an antibody comprises two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody" encompasses antigen-binding fragments of an antibody (e.g., single-chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv and domain antibody (dAb) fragments (de Wildt et al., Eur J Immunol. 1996; 26(3):629-39.)) as well as intact antibodies. Antibodies can possess the structural characteristics of IgA, IgG, IgE, IgD, and IgM (and their subclasses). Antibodies can be derived from any source, but primate (human or non-human primates) and primate-derived (primatized) antibodies are preferred.

[0083] In some embodiments, the antibodies described herein may be conjugated to a detectable label, and the binding of the detection reagent to the peptide of interest may be determined based on the intensity of the signal released from the detectable label. Alternatively, secondary antibodies specific to the detection reagent may be used. One or more antibodies may be conjugated to the detectable label. Any suitable label known in the art may be used in the assay methods described herein. In some embodiments, the detectable label comprises a fluorophore. As used herein, the term "fluorophore" (also referred to as "fluorescent label" or "fluorescent dye") refers to a moiety that absorbs light energy at a defined excitation wavelength and emits light energy at different wavelengths. In some embodiments, the detection moiety is an enzyme or contains an enzyme. In some embodiments, the enzyme is an enzyme that produces a colored product from a colorless substrate (e.g., β-galactosidase).

[0084] In other embodiments, the peptide of interest, as described herein, can be measured by liquid chromatography-mass spectrometry (LC-MS), an analytical technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry (MS).

[0085] Multiple reaction monitoring (MRM)-mass spectrometry is a highly sensitive and selective method for targeted quantification of protein / peptide abundance in complex biological samples. MRM-mass spectrometry is commonly used for the analysis of small molecules. Here, MRM enables the quantification of proteins in complex mixtures, providing a sensitive and selective tool for validating candidate biomarkers in disease processes. For this method, appropriate instruments such as the ABSciex 5500 or 6500QTrap mass spectrometer can be used, and the variability between various types of plasma samples can be evaluated after method optimization. Individual plasma samples are digested with appropriate proteases such as chymotrypsin or Glu-C to obtain HWMK or HWMK-tagged peptides for lysis as described herein.

[0086] The level (e.g., concentration) (e.g., expressed as AUC) of each tagged peptide can be determined using conventional methods. Where necessary, the ratio of the tagged peptide of cleaved HMWK relative to full-length HMWK to the tagged peptide of HMWK relative to LMWK can be calculated accordingly. Such ratios can be used to distinguish the presence of cleaved HMWK from full-length HMWK.

[0087] For example, the concentration of KHNLGHGH (SEQ ID NO:1), a tag peptide representing the 46 kDa light chain of 2-chain HMWK produced by chymotrypsin digestion, has been found to be significantly higher in plasma samples from HAE patients than in plasma samples from healthy human subjects. These results suggest that the 46 kDa light chain of 2-chain HMWK represented by this tag peptide is a reliable biomarker for the diagnosis and prognosis of HAE.

[0088] In another instance, the target peptides present in plasma in this study were found to be attributable to HMWK, as all peptides including HMWK and LMWK-tagged peptides (SYYFDLTDGLS (SEQ ID NO:10)) were confirmed by MRM-based HMWK standards and samples digested for Glu C. A significant increase in SSRIGE (SEQ ID NO:5) peptide was observed in HAE SCAT plasma compared to normal SCAT plasma, and an increase was also observed between FXIIa-activated and unactivated plasma, where HMWK peptides remained unchanged. A higher SSRIGE (SEQ ID NO:5) / SYYFDLTDGLS (SEQ ID NO:10) ratio was also found in HAE SCAT plasma samples compared to normal SCAT plasma samples, and this ratio was also higher in activated plasma compared to unactivated plasma. The relative abundance ratios of SSRIGE (SEQ ID NO:5) and SYYFDLTDGLS (SEQ ID NO:10) were 4.4 and 8.9 for normal SCAT plasma and HAE SCAT plasma, respectively, and 19.02 and 48.63 for normal citrate plasma and FXIIa activated citrate plasma, respectively.

[0089] Plasma samples lacking kininogen (negative control) showed a low-intensity peak near the detection limit of the SSRIGE (SEQ ID NO:5) peptide, which is expected to be due to the presence of LMWK. The HMWK long peptide KKIYPTVNCQPLGMISLMKRPPGFSPFRSSRIGE (SEQ ID NO:18) was also measured in plasma samples using a targeted assay (MRM). The HMWK long peptide showed little change between normal SCAT and HAE SCAT plasma samples, and between activated and unactivated samples.

[0090] II. Reagent Kit

[0091] This disclosure also provides kits for measuring the level of a tagged peptide in lysed HMWK and / or for distinguishing between lysed HMWK and full-length HMWK in a sample, such as a biological sample from a human patient. Such kits may contain a tagged peptide-specific assay (e.g., chymotrypsin or GluC); a tagged peptide-specific assay produced by digestion with a suitable protease; a vacuum-sealed blood collection tube; and, optionally, lysed kininogen standards and / or intact kininogen standards as controls. In some embodiments, the kit further includes a secondary antibody and / or reagents for detecting the binding of the assay to the peptide of interest.

[0092] In some implementations, the kit may include instructions for use according to any of the methods described herein. These instructions may include a description of how to use the components contained in the kit for measuring the level of the tagged peptide in a sample treated with a protease.

[0093] Instructions for use of the kit typically include information about the amounts of the various components and appropriate conditions for performing the assays described herein. Instructions for use provided with the kits of this invention are typically in text form on a label or packaging instruction (e.g., on paper included in the kit), but machine-readable instructions (e.g., instructions carried on magnetic and optical storage disks) are also acceptable.

[0094] The label or package insert indicates that the kit is used to measure the level of the tagged peptide in cleaved HMWK and / or to distinguish between cleaved HMWK and full-length HMWK. Instructions for use are provided for practicing any of the methods described herein.

[0095] The kit of the present invention is packaged in appropriate packaging. Appropriate packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed polyester film (Mylar) or plastic bags), etc.

[0096] The kit may optionally include additional components such as buffers and explanatory information. Typically, the kit includes a container and a label on or associated with the container, or one or more instruction manuals. In some embodiments, this disclosure provides an article of manufacture containing the contents of the kit described above.

[0097] III. Application of the determination method

[0098] (i) Clinical applications: diagnosis and prognosis of diseases

[0099] Approximately 75–90% of circulating prokallikrein binds to HMWK via interaction with its inactive site and domain 6 of HMWK. Free and HMWK-bound active pKal produces cleaved HMWK and bradykinin. The applicability of these biomarkers can be demonstrated by tracking their levels in the presence or absence of acute HAE episodes. The levels of these biomarkers can also be altered during bradykinin-mediated edema or other episodes mediated by pKal activity.

[0100] The assay methods and kits described herein can be applied to the assessment of diseases, such as disease diagnosis or prognosis. Assessment may include identifying subjects at risk of or suffering from diseases as described herein, such as pKal-mediated disorders like HAE. Assessment also includes monitoring treatment for the disease, such as evaluating the effectiveness of treatment for pKal-mediated disorders (e.g., HAE). Additionally, assessment may include identifying diseases that can be treated with pKal inhibitors.

[0101] A. diagnosis

[0102] In some embodiments, assay methods and kits are performed to determine the levels of cleaved kininogen and / or intact kininogen in biological samples (e.g., blood or plasma samples) collected from candidate subjects (e.g., human patients suspected of having PKal-mediated disorders, such as HAE). The levels of cleaved HMWK (e.g., 2-chain HMWK or its heavy and / or light chains, including 56 kDa and 46 kDa light chains) and / or the ratio between the levels of cleaved HMWK and intact HMWK can be determined based on the levels of tagged peptides of cleaved HMWK as described herein and / or the ratio between tagged peptides of cleaved HMWK and tagged peptides of HMWK (e.g., relative to LMWK). The concentrations or ratios of such tagged peptides can then be compared with predetermined reference values ​​or reference ratios to determine whether the subject has a PKal-mediated disorder (such as HAE) or is at risk of having a PKal-mediated disorder (such as HAE). For example, if the concentration of the tagged peptide or the ratio of the two tagged peptides in a candidate subject's sample is at or above the reference value / reference ratio, the subject may be identified as having or at risk of having a PKal-mediated disorder (such as HAE).

[0103] The reference value / reference ratio can be a control level of the tagged peptide as described herein or a ratio of two tagged peptides. In some embodiments, the control value / control ratio represents the value / ratio of the tagged peptide in a control sample, such as a sample (e.g., a blood or plasma sample) obtained from a healthy subject or a population of healthy subjects (preferably of the same species as the candidate subjects). As used herein, a healthy subject is a subject who clearly does not have the target disease (e.g., PKal-mediated disorder such as HAE) or has no history of said disease when the levels of cleaved and / or intact kininogen are measured.

[0104] In some implementations, control samples can be obtained from human HAE patients in the disease resting phase. The elevated level of cleaved HMWK-tagged peptides, or the elevated ratio between cleaved HMWK-tagged peptides and HMWK-tagged peptides, relative to a reference value / reference ratio obtained from such control samples, can indicate the risk of HAE onset.

[0105] Reference values / reference ratios can also be predetermined values ​​or ratios. Such predetermined values / ratios can represent the value of a cleaved HMWK-tagged peptide (e.g., a 46 kDa light chain-tagged peptide of 2-chain HMWK) or the ratio of two tagged peptides as described herein in a population of subjects who do not have the target disease or are not at risk for the target disease. It can also represent the value (e.g., concentration) of a cleaved HMWK-tagged peptide (e.g., a 46 kDa light chain-tagged peptide) or the ratio of two tagged peptides as described herein in a population of subjects who have the target disease (e.g., are in a disease resting phase).

[0106] The predetermined value / ratio can take many forms. For example, it can be a single cutoff value, such as a median or average. In some implementations, this predetermined level can be established based on comparison groups, such as a defined group known to have the target disease and another defined group known not to have the target disease. Alternatively, the predetermined level can be a range, for example, representing a range of ratios of the two peptides of interest in a control population within a predetermined percentage range.

[0107] The control values / ratios described herein can be determined using conventional techniques. In some instances, control values / ratios can be obtained by performing conventional methods on control samples also as described herein (e.g., the same methods used to determine the levels of the two peptides of interest in the test samples as described herein). In other instances, the levels of the tagged peptides of interest can be obtained from members of a control population, and the results can be analyzed using computational procedures to obtain control levels (predetermined levels) representing the levels of cleaved kininogen and / or intact kininogen in the control population.

[0108] By comparing the concentration of the HMWK-tagged peptide cleaved as described herein, or the ratio of two tags of interest as described herein, in a sample obtained from a candidate subject with a reference ratio as described herein, it can be determined whether the candidate subject has a PKal-mediated disease (e.g., HAE) or is at risk of a PKal-mediated disease, or whether a patient with HAE is at risk of an HAE flare-up. For example, if the value of the HMWK-tagged peptide cleaved in a candidate subject's sample or the ratio of two tags of interest deviates from a reference value or reference ratio (e.g., increases compared to a reference value or reference ratio), the candidate subject may be identified as having the disease or being at risk of the disease, or a patient with HAE is at risk of an HAE flare-up. When a reference value or reference ratio represents a range of values ​​or ratios of tags of interest as described herein in a population of subjects with the target disease, a value or ratio of the tags of interest in a candidate's sample falling within that range indicates that the candidate subject has the target disease or is at risk of the target disease.

[0109] As used herein, “elevated value / ratio or value / ratio above reference value / reference ratio” means that the value of the labeled peptide or the ratio of two labeled peptides is higher than a reference value or reference ratio, such as a predetermined critical ratio of the same labeled peptide or the same two labeled peptides in a control sample. Control levels are described in detail herein. Elevated values ​​of the labeled peptide of interest or elevated ratios of two labeled peptides of interest include values / ratios such as 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500% or higher of the reference value / reference ratio.

[0110] As used herein, “a reduced value / ratio below a reference value / reference ratio” refers to a level of the tagged peptide of interest or a ratio of two tagged peptides of interest that is lower than a reference value / reference ratio, such as a predetermined threshold value for the same tagged peptide of interest or two tagged peptides of interest in a control sample. Control levels are described in detail herein. Reduced values ​​of tagged peptides or ratios of two tagged peptides include values / ratios that are, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, or more lower than a reference ratio of two tagged peptides of interest.

[0111] In some embodiments, the candidate subject is a human patient with symptoms of a pKal-mediated disorder (e.g., HAE). For example, the subject has edema, swelling, said swelling being entirely or primarily peripheral; urticaria; swelling with redness, pain, and no signs of infection; nonhistamine-mediated edema, recurrent episodes of swelling, or a combination thereof. In other embodiments, the subject did not have symptoms of a pKal-mediated disorder, a history of pKal-mediated disorder symptoms, or a history of a pKal-mediated disorder, such as HAE, at the time of sample collection. In other embodiments, the subject is resistant to antihistamine therapy, corticosteroid therapy, or both antihistamine therapy and corticosteroid therapy.

[0112] B. Assess treatment effectiveness

[0113] The assays described herein can also be applied to assess the effectiveness of treatment for pKal-mediated disorders (e.g., HAE). For example, various biological samples (e.g., blood or plasma samples) can be collected from the subject, which may be processed before and after treatment or during treatment. The level of the tagged peptide can be determined by any of the assays described herein, and the level of the tagged peptide of cleaved HMWK (e.g., 46 kDa light chain) or the ratio of cleaved -HMWK-specific peptides to HMWK-specific peptides can be determined accordingly. If the value of the tagged peptide of cleaved HMWK (e.g., for the 46 kDa light chain) or the ratio of the two tagged peptides decreases after or during treatment (the level of the tagged peptide of cleaved HMWK (e.g., 46 kDa light chain) or the ratio of the two tagged peptides of interest in a later-collected sample compared to its value in a previously collected sample), this indicates that the treatment is effective. In some instances, treatment involves therapeutic agents such as kallikrein binders, bradykinin B2 receptor antagonists, or C1-INH substitutes as described herein. Examples of therapeutic agents include, but are not limited to, DX-2930, SHP643, or DX88.

[0114] If a subject is identified as unresponsive to treatment, a higher dose and / or a higher frequency of administration of the therapeutic agent is administered to the identified subject. In some embodiments, the dose or frequency of administration of the therapeutic agent is maintained, reduced, or discontinued in subjects identified as responsive to treatment or who do not require further treatment. Alternatively, different treatments may be applied to subjects found unresponsive to the first treatment.

[0115] (ii) Non-clinical applications

[0116] Furthermore, the assay methods described herein have non-clinical applications, such as for research purposes and / or preclinical drug development purposes. While many diseases associated with pKal have been identified, it is possible that other diseases are mediated by similar mechanisms or involve similar components. In some embodiments, the methods described herein can be used to identify diseases associated with pKal. In some embodiments, the methods described herein can be used to study the mechanisms of a disease (e.g., to discover novel biological pathways or processes in the development of a disease) or disease progression.

[0117] In some implementations, the development of novel therapeutics for pKal-related diseases may rely on the levels or ratios of tagged peptides measured using the assay methods described herein. For example, the levels or ratios of tagged peptides as described herein can be measured from samples obtained from subjects who have received the novel therapeutic (e.g., in clinical trials) or from samples obtained from in vitro studies. In some implementations, the levels or ratios of tagged peptides may indicate the activity of the novel therapeutic in in vitro studies or the efficacy of the novel therapeutic in a clinical trial setting.

[0118] Without further explanation, it is believed that those skilled in the art can fully utilize this disclosure based on the foregoing description. Therefore, the specific embodiments described below are merely illustrative and do not limit the other parts of this disclosure in any way. All publications cited herein are incorporated by reference for the purposes of this document.

[0119] Example

[0120] Example 1: Methods for distinguishing between cleaved high molecular weight kininogen (HMWK) and full-length HMWK

[0121] The overall objective of this embodiment is to develop a robust method for LC-MS-based peptide quantification to distinguish between full-length kininogen (HK; single-chain or 1-chain HK) and kininogen cleaved by kallikrein (two-chain products or 2-chain HK, and the heavy and / or light chains of the two-chain products). In some embodiments, this semi-quantitative assay measures the ratio of 2-chain HK product to 1-chain HK substrate to determine the cut-point of 2-chain HK in biological samples (e.g., plasma) from healthy volunteers compared to patients with diseases associated with plasma kallikrein, such as HAE.

[0122] Single-chain HMWK (1-chain HK) and two-chain HMWK (2HK) (Enzyme Research Laboratories and Sigma) were used as standards in the examples described below. Different types of plasma samples (citrate, citrate + protease inhibitor (PI), normal SCAT plasma (plasma samples collected from healthy subjects in SCAT tubes), and HAESCAT plasma (plasma samples collected from HAE patients in SCAT tubes)) were used in the examples described below. See, for example, PCT / US2016 / 046681, the related disclosure of which is incorporated herein by reference. Quality control was performed between runs, and reproducibility checks were conducted to address variability across different days and runs. EDTA-plasma (Biochem Services) was used as a control for method optimization.

[0123] The assay described and developed in this embodiment provides a sensitive and selective method for validating candidate biomarkers in disease processes. For this method, an ABSciex 5500 or 6500Qtrap mass spectrometer was used, and variability between different types of plasma samples was assessed after method optimization. Individual plasma samples (including normal SCAT plasma and HAE SCAT plasma, kininogen-deficient plasma, normal citrate plasma, factor XIIa-activated normal citrate plasma, and HK standards) were digested with Glu C, and multiple reaction monitoring (MRM) analysis was performed on the peptides of interest. Glu C was used as an exemplary protease to obtain peptides specific to distinguish between 1-chain HK and 2-chain HK (tagged peptides). Briefly, the initial experiments were designed to validate the quality of protein standards for specific peptides using MRM mode. 10 μl samples containing 10 μg of protein were added to a C18 column. Precursor and protease digestion product ions specific to glut-C-digested plasma kininogen were selected using Skyline software. For plasma kininogen, target candidate peptides SSRIGE (SEQ ID NO:5), SSRIGEIKE mis 2 (SEQ ID NO:19), KKIYPTVNCQPLGMISLMKRPPGFSPFRSSRIGE (SEQ ID NO:20), KKIYPTVNCQPLGMISLMK (SEQ ID NO:21), and SYYFDLTDGLS (SEQ ID NO:10), along with 3-5 product ions, were empirically validated. After performing MRM using a skyline-guided method, the fragmentation and collision energies of the peptides were individually optimized. Data were analyzed using Analyst Software (ABSciex). The development of this method focused on optimizing sample preparation, enzymatic digestion for different types of plasma samples and HK standards, chromatographic analysis, and mass spectrometry parameters.

[0124] (i) Immunoprecipitation samples and whole plasma samples

[0125] To validate the validation concept, four protein samples (IP samples) and four whole plasma samples (non-IP samples) obtained via immunoprecipitation were used in the study. Samples were eluted with SDS followed by removal using a cleaning agent removal column. Targeted assays were performed on an ABSciex 5500Qtrap triple quadrupole mass spectrometer to examine peptides of interest in the samples.

[0126] All four peptides of interest, SSRIGE (SEQ ID NO:5), SSRIGEIKE (SEQ ID NO:19), KKIYPTVNC QPLGMISLMKRPPGFSPFRSSRIGE (SEQ ID NO:18), and KKIYPTVNCQPLGMISLMK (SEQ ID NO:21), were found in non-IP samples but not in IP plasma samples. No peak differences were found between activated and unactivated plasma samples (treated with FXIIA). Table 2 below shows the peak intensities of the peptides in IP and non-IP samples. No difference in peak intensity was observed between activated and unactivated samples for SSRIGE (a specific peptide derived from the 2HK light chain via Glu C digestion; SEQ ID NO:22) or KKIYPTVNC QPLGMISLMKRPPGFSPFRSSRIGE (a specific peptide derived from the HK long peptide via Glu C digestion; SEQ ID NO:18) (indicated by the rows in the boxes).

[0127] EDTA plasma was run as a positive control alongside IP / non-IP samples, and all peptides of interest were found in both types of samples.

[0128] Table 2: Peak intensities of five peptides of interest in IP and non-IP samples

[0129]

[0130] The results showed that whole plasma samples digested with Glu C provided sufficient sensitivity to detect both 1-chain and 2-chain HK peptides. Therefore, whole plasma samples digested with the exemplary Glu C protease were used in the experiments described below.

[0131] (ii) Sample preparation optimization

[0132] (a) Digestion plan

[0133] To optimize plasma GluC digestion, various protocols were employed. The different parameters modified in these protocols are summarized below:

[0134] Option 1: Add a denaturation step before digestion (using a solution such as 0.1M DTT).

[0135] - Option 2: Use different concentrations of reducing agent (0.05M DTT)

[0136] - Option 3: Dilute the sample 10-fold before digestion; and

[0137] Method 4: Increase the enzyme incubation time to 19 hours.

[0138] Based on peak intensity and peak shape, Method 2 can better detect SSRIGE (SEQ ID NO:5) and KKIYPTVNCQPLGMISLMK (SEQ ID NO:21). Therefore, the digestion method of Method 2 was used in subsequent experiments.

[0139] (b) reduction temperature and incubation time

[0140] The reduction temperature and incubation time were also optimized. In short, HK standards and EDTA-plasma were used to evaluate the temperature (55°C vs. 90°C) and incubation time (30 mins vs. 60 mins) for reducing the sample during digestion.

[0141] -HK: 90℃ for 1 hour

[0142] -HK: 55℃ for 30 minutes

[0143] - Plasma EDTA (Biochem): 90℃ for 1 hour

[0144] - Plasma EDTA (Biochem): 55℃ for 30 minutes

[0145] Samples were analyzed using multiple reaction monitoring (MRM) to detect the peptides SSRIGE (SEQ ID NO: 5) and KKIYPTVNCQPLGMISLMKRPPGFSPFRSSRIGE (SEQ ID NO: 20). A higher peak intensity was found in this sample after reduction at 90°C for 1 hour compared to reduction at 55°C for 30 minutes for both HK standard and plasma samples. The reaction conditions, including incubation at 90°C for 1 hour, were used to reduce the sample in subsequent experiments.

[0146] (c) Plasma protease inhibitors (PIs) and anticoagulants

[0147] The effects of the presence of PI and anticoagulants in plasma on digestive efficiency were also evaluated. Digestive efficiency was assessed for plasma with and without PI, and also for plasma containing citrate as an anticoagulant versus plasma containing EDTA. Samples were evaluated using an AB Sciex 5500Qtrap LC-MS / MS system by MRM analysis, and peak intensities were compared for SSRIGE (SEQ ID NO:5). Figure 3 As indicated by the middle arrow. These results show that:

[0148] EDTA plasma without PI > EDTA plasma with PI > citrate plasma without PI > citrate plasma with PI.

[0149] These results also indicate that citrate and protease inhibitors have a negative impact on Glu C digestion, as reduced peptide responses were observed in samples containing citrate, PI, or both.

[0150] (iii) Different enzyme digestions used to check sequence coverage

[0151] To examine whether sequence coverage was enzyme-dependent, different enzymes and trypsin were used. HK standard samples from Sigma and EDTA-plasma samples from Biochem were digested with trypsin and run on a high-resolution precision mass instrument (HRAM), and analyzed using Proteome Discoverer. TM Software analysis results.

[0152] Both HK samples and plasma samples digested with trypsin showed good sequence coverage of the full-length kininogen.

[0153] (iv) EDTA-SIM (Single Ion Monitoring) in plasma and HK samples

[0154] High concentrations of HK and plasma samples were used for SIM, and the peptides of interest were detected using a high-resolution precision mass spectrometry (HRAM) instrument. SIM-MS was performed on Sigma samples of EDTA plasma and HK. 2 Data analysis.

[0155] Analysis of Glu C-digested EDTA plasma samples on the HRAM instrument revealed the detection of all four peptides: SSRIGE (SEQ ID NO:5), KKIYPTVNCQPLGMISLMK (SEQ ID NO:21), long HK peptide, and bradykinin. Regarding the HK standard sample, the peptides SSRIGE (SEQ ID NO:5), KKIYPTVNCQPLGMISLMK (SEQ ID NO:21), and bradykinin were found, but long HK peptide was not detected.

[0156] To confirm the consistency of the standards, HK from two different sources (Enzyme Research and Sigma) was used. However, no detectable differences were observed between the two standards in this experiment, and the long HK peptide was not detected in either standard sample.

[0157] (v) Different types of plasma, different Glu C and different enzyme-to-protein ratios

[0158] To further optimize GluC digestion, different types of plasma samples (EDTA, citrate, containing PI, and without PI), different GluC sources (Protea & Promega), and different enzyme:protein ratios (1:20 and 1:10) were evaluated. Furthermore, various detection platforms, including full scan, SIM-MS2 (on HRAM), and MRM (QQQ; a ternary quadrupole instrument), were used to detect peptides of interest in different samples. Table 3 summarizes the various combinations of sample types, enzymes, and dilutions used in this experiment.

[0159] Table 3: Summary of Test Conditions

[0160] EDTA plasma (Biochem) Protea Glu C and Promega Glu C 1:20 and 1:10 Citrate-treated plasma (Sponsor) Protea Glu C and Promega Glu C 1:20 and 1:10 HK Sigma Protea·Glu C and Promega Glu C 1:20 and 1:10 EDTA plasma (Biochem) + PI Protea Glu C and Promega Glu C 1:20 citrate plasma (Sponsor) + PI Protea Glu C and Promega Glu C 1:20

[0161] (a) Full scan and SIM-MS 2 (HRAM)

[0162] Different types of plasma and standard samples, as described above, were digested with Glu C, and the digestion products were run on an HRAM instrument for full scan and SIM-MS. 2 Sample information is shown on the right-hand side of the color illustration. (Using ProteomeDiscoverer) TM Software and Xcalibur TM The software was used to analyze and determine the peak intensity of each peptide of interest. For plasma samples, the following peptide intensities were observed for SSRIGE (SEQ ID NO:5):

[0163] EDTA plasma without PI > EDTA plasma with PI > citrate plasma without PI > citrate plasma with PI.

[0164] A 1:20 (enzyme:protein) ratio was determined to result in a better response than a 1:10 (enzyme:protein) ratio. For the long HK peptide, the intensity was only minimally affected by plasma type or enzyme:protein ratio. The long HK peptide was not observed in any standard samples; however, KKYIPTVNCQPLGMISLMK peptide (SEQ ID NO:23) and bradykinin were detected in all standards, suggesting that the long HK peptide may be degraded. This can be explained by the observation of various fragments of the long HK peptide, including KKIYPTVNCQPLGMISLMK (SEQ ID NO:21), RPGFSPFR (SEQ ID NO:24), and SSRIGE (SEQ ID NO:5), without being bound by any particular theory.

[0165] Table 4 below shows the use of full scan and SIM-MS on HRAM instruments. 2 The data represents the intensity of the peptides of interest (classified by sample type).

[0166] Table 4: Intensity of peptides of interest

[0167]

[0168]

[0169] ++++: High abundance

[0170] +++: Medium abundance

[0171] ++: Low abundance

[0172] +: Very low abundance

[0173] + / -: Negligible abundance

[0174] -: Does not exist

[0175] (b) Multiple Response Monitoring (MRM) Targeted Assay

[0176] Using the same series of samples as described above, the peptides of interest were detected using the targeted assay (MRM). After detection using an ABSciex Qtrap 5500 instrument, the samples were analyzed using AB SciexAnalyst software. Sample information is displayed on the right-hand side of the color plot.

[0177] Regardless of sample type and conditions, the detection of SSRIGE peptide (SEQ ID NO:5) was consistently observed. With optimized sample preparation, SSRIGEKE (SEQ ID NO:25) peptide was not detected. However, the long HK peptide was present only in plasma samples and not in HK standard samples. KKYIPTVNCQPLGMISLMK (SEQ ID NO:23) peptide and bradykinin were present in all HK standard samples, but the long HK peptide was not detected. These results are consistent with those described above from full scan and MS. 2 The datasets are similar and suggest that the HK long peptide may be degraded, further explaining the observed HK long peptide fragments shown above.

[0178] Table 5 below shows the intensity of peptides of interest (categorized by sample list) using MRM data from an AB Sciex Qtrap 5500 instrument.

[0179] Table 5: Intensity of peptides of interest

[0180]

[0181]

[0182] ++++: High abundance

[0183] +++: Medium abundance

[0184] ++: Low abundance

[0185] +: Very low abundance

[0186] + / -: Negligible abundance

[0187] -: Does not exist

[0188] (c) Sequence Coverage Using HRAM and Proteome Discoverer™ Software

[0189] To determine the optimal Glu C concentration for protein digestion, sequence coverage of HK standard samples was evaluated by full scan on an HRAM instrument after digestion with Glu C (Protea and Promega) from different sources and different enzyme:protein concentrations (1:20 and 1:10). Optimal digestion was achieved with a 1:20 dilution of Glu C from any source due to higher sequence coverage in the region of interest. As provided below, a 1:10 dilution also provides coverage in the region of interest, but with lower confidence scores for peptides.

[0190]

[0191] In the amino acid sequence of the 1-chain HMWK above, peptides derived from Glu-C, shown in bold, were identified using single-ion identification, while peptides indicated by underline were identified using multi-ion identification. Brachykinin peptides are indicated in bold italics.

[0192] (vi) Sequence alignment of high molecular weight bradykinin (HMWK) and low molecular weight bradykinin (LMWK)

[0193] To identify peptides specific to HMWK compared to LMWK, sequence alignment of these two types of bradykinin was performed using UniProt software provided below (hHMWK: SEQ ID NO: 11; hLMWK: SEQ ID NO: 12):

[0194]

[0195] Compared to LMWK, four peptides were found to be specific to HMWK:

[0196] 1. SYYFDLTDGLS (SEQ ID NO:10)

[0197] 2. INPTTQMKE (SEQ ID NO:26)

[0198] 3. KQRKHNLGHGHKHE (SEQ ID NO:7)

[0199] 4. EDSTTPSAQTQE (SEQ ID NO:27)

[0200] a. Using full scan and MS on HRAM 2 Detection of HMWK-specific peptides in HK samples

[0201] Starting with the specific peptide SYYFDLTDGLS (SEQ ID NO:10), a full scan and MS were performed using Glu C-digested HK standards. 2 Full scan and MS 2 Run on the HRAM instrument and use Proteome Discoverer TM Software analysis. As shown below, a peptide specific to HMWK was detected in the HK standard, suggesting that the HK standard contains HMWK.

[0202]

[0203] In the HMWK amino acid sequence shown above, the positions of the HMWK-specific peptides are indicated by bold and underline.

[0204] b. HMWK-specific peptides were detected in HK samples and EDTA-treated plasma using MRM.

[0205] To determine the presence of HMWK in the standards and compare them with plasma samples, Glu C-digested HK and Glu C-digested plasma were run on an ABSciex 5500Qtrap for targeted analysis. HMWK-specific peptides (SYYFDLTDGLS; SEQ ID NO:10) were detected in both samples using the MRM assay; however, the peak intensity of the peptides was lower in plasma compared to the HK standards. This is predictable due to the complexity and high background noise of plasma. These results confirm that these peptides are derived from HMWK.

[0206] (vii) Digestive conditions

[0207] a. HK and plasma were digested with Glu C at 25°C and 37°C.

[0208] The effect of digesting HK and plasma samples with Glu C at 25°C instead of 37°C was evaluated. HMWK-specific peptides were detected in the HK standards, but long HK peptides were not detected in the standards. To test for incomplete digestion or whether high temperature degrades the HK standards into bradykinin and KKYIPTVNCQPLGMISLMK (specific to the 2HK heavy peptide; SEQ ID NO: 23), plasma and HK samples were digested with Glu C at 25°C instead of 37°C. MRM analysis was used to target and detect peptides of interest, including HMWK-specific peptides. Peak intensities are as follows:

[0209] HK Sigma Glu C 25℃:

[0210] ●SSRIGE (SEQ ID NO:5): 5960

[0211] ●KKIYPTVNCQPLGMISLMKRPPGFSPFRSSRIGE(SEQ ID NO:20):4000

[0212] ●SYYFDLTDGLS(SEQ ID NO:10):7.6e4

[0213] HK Enzyme Research (Glu C 25℃):

[0214] ●SSRIGE(SEQ ID NO:5):4.5e5

[0215] ●SSRIGEIKE mis 2 (SEQ ID NO:19): 1.5e5

[0216] ●KKIYPTVNCQPLGMISLMKRPPGFSPFRSSRIGE(SEQ ID NO:20):2.3e5

[0217] ●SYYFDLTDGLS(SEQ ID NO:10):4.6e5

[0218] The study found that digestion of HK standards (from Enzyme Research and Sigma) at 25°C with Glu C resulted in the detection of all three peptides of interest, including the long HK peptide, indicating that the standards were degraded at high temperatures and therefore the long HK peptide was not observed upon digestion at 37°C. The standards from Enzyme Research produced higher peak intensities compared to those from Sigma. The mis2-cleaved peptide SSRIGEIKE (SEQ ID NO:19) was found only in the Enzyme Research standards. Regarding plasma samples, the results indicated that digestion with Glu C at 37°C resulted in better digestion than digestion with Glu C at 25°C. Figure 1 Using standards at 25°C confirmed that the long peptide and other HMWK-specific peptides indicated that the peptides observed in the plasma samples originated from HK.

[0219] No peptides other than SSRIGE (SEQ ID NO:5) were detected in the plasma sample at 25°C; however, as expected, all peptides of interest were observed in the plasma sample digested at 37°C. Figure 1 HK long peptides were observed in both standards at 25°C instead of 37°C.

[0220] These results suggest that Glu C digestion is better for standard samples at 25°C, while digestion of plasma is better at 37°C.

[0221] a. Optimized digestion method using high-resolution precision mass (HRAM) instruments

[0222] Samples digested at 25°C and 37°C with Glu C- were also fully scanned and evaluated on the HRAM instrument to detect peptides of interest, including long peptides of HK and peptides specific to HMWK, thereby determining whether another detection platform could be used to detect peptides of interest in plasma samples digested at 25°C.

[0223] All peptides of interest and HMWK-specific peptides were detected in HK standard samples digested at 25°C and plasma samples digested at 37°C. This dataset is consistent with the MRM data described above, except for the mis2 cleaved peptide SSRIGEIKE (SEQ ID NO:19), which is commonly seen in HK standards from Enzyme Research. The LMWK-specific peptide YKGRPPKAGAE (SEQ ID NO:28) was also included in this assay and was found in plasma samples rather than HK standards, which is consistent with expectations. Figure 2 , 3 ).

[0224] b. HK – Full scan sequence coverage at 25°C

[0225] Sequence coverage of HK standards was evaluated using optimized HK Glu C digestion, Glu C source, and enzyme-to-protein ratio. In summary, Glu C digestion at 25°C with an enzyme-to-protein ratio of 1:20 resulted in significantly improved sequence coverage of HK (derived from both Enzyme Research and Sigma), with high confidence scores, particularly in the regions of interest (long HK peptides).

[0226]

[0227] In the amino acid sequence of HMWK shown above, the positions of the long HK peptides are indicated by bold and underline.

[0228] c. Full scan and SIM on HRAM instruments

[0229] To detect long HK peptides, HK standards digested at 25°C were subjected to SIM and full scan using an HRAM instrument. Following digestion at 25°C, long HK peptides were observed in the HK standards via full scan and SIM on the HRAM instrument.

[0230] (viii) For HMWK and LMWK-specific peptides digested with Glu C at 25°C

[0231] Two additional HMWK-specific peptides (as opposed to LMWK), INPTQMKE (SEQ ID NO:29) and SYYDDGLS (SEQ ID NO:30), were added, and MRM analysis was performed to detect these specific peptides. After digestion of HK standards (HKSigma and HK Enzyme Research) at 25°C, the additional HMWK-specific peptides were observed, confirming the presence of HK.

[0232] (ix) Use optimized methods to evaluate the differences between different types of samples.

[0233] a. Quantification of SSRIGE (SEQ ID NO) in plasma samples using the targeted assay (MRM) NO:5)

[0234] Individual plasma samples were digested with Glu C and subjected to MRM analysis to detect the peptide of interest SSRIGE (SEQ ID NO: 5). Sample types included normal SCAT and HAESCAT plasma samples, kininogen-deficient plasma samples, normal citrate plasma samples, factor XIIa-activated normal citrate plasma samples, and HK standard samples.

[0235] like Figure 3 The results show that the SSRIGE (SEQ ID NO:5) peptide was quantified using the Analyst software's quantitative procedure. A significant increase in SSRIGE (SEQ ID NO:5) peptide was detected in HAE SCAT plasma samples compared to normal SCAT plasma samples. A significant increase in SSRIGE (SEQ ID NO:5) peptide was also present in activated plasma compared to unactivated plasma samples. Plasma samples lacking kininogen showed minimal amounts of SSRIGE (SEQ ID NO:5) peptide, as expected due to the absence of HMWK.

[0236] b. Quantification of HK long peptides in plasma samples using the targeted assay (MRM)

[0237] Plasma samples were digested with Glu C and subjected to MRM analysis to detect long HK peptides. Sample types included normal SCAT and HAE SCAT plasma samples, kininogen-deficient plasma samples, normal citrate plasma samples, factor XIIa-activated normal citrate plasma samples, and HK standards. Extremely minimal variations in the amount of long HK peptides detected were observed between normal SCAT and HAE SCAT plasma samples. Furthermore, no difference was observed between activated and inactivated samples. Figure 4 .

[0238] (x) Assess the consistency and reproducibility of Glu C digestion.

[0239] To assess the reproducibility of day-to-day Glu C digestion, HK standard and plasma (plasma citrate + PI) samples were digested for three consecutive days, and MRM analysis was performed to detect peptides of interest (see Tables 6A and 6B below). All peptides of interest were present in both the HK standard and plasma samples, exhibiting the indicated peak intensities. Tables 6A and 6B show consistent results for Glu C digestion data for both HK standard and plasma (plasma citrate + PI) over the three days.

[0240] Table 6A: Digestion of HK and Glu C for three consecutive days

[0241]

[0242] Table 6B: Digestion of plasma citrate + PI and Glu C for three consecutive days

[0243]

[0244] The ratio between (xi)SSRIGE (SEQ ID NO:5) and SYYFDLTDGLS (SEQ ID NO:10)

[0245] a. Calculate SSRIGE (SEQ ID NO:5) and SYYFDLTDGLS peptide (SEQ ID NO:5). The ratio of NO:10)

[0246] The ratio between the SSRIGE (SEQ ID NO:5) and SYYFDLTDGLS (SEQ ID NO:10) peptides was calculated using a targeted assay (MRM) in normal plasma and plasma from individuals with HAE, employing Glu C-digested HK and plasma samples. Measuring the ratio of the SSRIGE (SEQ ID NO:5) peptide to HMWK-specific peptides can, for example, be used in diagnostic assays. The software quantifies the area under the peak of the peptide to calculate the total ratio and the average ratio.

[0247] Compared to normal SCAT plasma samples, an overall increase in the SRIGE (SEQ ID NO:5) / SYYFDLTDGLS (SEQ ID NO:10) ratio was observed in HAE SCAT plasma samples. For unactivated plasma, the SRIGE (SEQ ID NO:5) / SYYFDLTDGLS (SEQ ID NO:10) ratio was also significantly increased in activated plasma. Figure 5 ).

[0248] b. The average ratio between SSRIGE (SEQ ID NO:5) and SYYFDLTDGLS peptide (SEQ ID NO:10)

[0249] The average ratio of SSRIGE (SEQ ID NO:5) / SYYFDLTDGLS (SEQ ID NO:10) calculated using the MRM targeted assay across different sample types is the area under the peak ( Figure 6 Compared to normal SCAT plasma samples, a higher mean ratio of SSRIGE (SEQ ID NO:5) / SYYFDLTDGLS (SEQ ID NO:10) was found in HAE SCAT plasma samples. This ratio of the SSRIGE (SEQ ID NO:5) peptide relative to the HMWK and LMWK-specific peptide SYYFDLTDGLS (SEQ ID NO:10) has the potential to be used in assays, for example, for diagnosing diseases or disorders associated with cleaved kinins or for differentiating between samples from healthy and diseased individuals.

[0250] (xii) LC-MS operation optimization using HK standard products

[0251] To enhance the method's effectiveness, LC-MS execution was optimized by improving gradients and reducing runtime. HK standards were primarily used for LC-MS optimization. Figure 6 As shown, results for several methods are presented from various LC-gradients and run times used. The LC-MS method successfully reduced the run time to 10 min. The chromatogram in the lower left corner shows the "optimized method" when the SYYFDLTDGLS peptide (SEQ ID NO:10) appears as a single peak instead of a split peak. Significantly reduced run times were found for the two peptides SSRIGE (SEQ ID NO:5) and SYYFDLTDGLS (SEQ ID NO:10), which are essential for ratio calculation.

[0252] (xiii) Optimization of LC-MS operation using plasma samples

[0253] The shorter LC-MS run described above was validated using plasma samples, in which the peptides of interest (SSRIGE (SEQ ID NO:5) and SYYFDLTDGLS (SEQ ID NO:10)) were detected. The method, with a run of 16.5 min, detected the peptides of interest in both HK standards and plasma samples, representing a significant improvement over the original method (42 min). The intensity of the two peptides decreased with increasing plasma sample complexity: EDTA plasma > citrate plasma > SCAT plasma.

[0254] Example 2: Quantification of the 46 kDa light chain of cleaved HWMK in HAE plasma using tagged peptides and LC-MS / MS

[0255] The exemplary method described in this embodiment is designed to determine the concentration of endogenous 2-chain HMWK (which can be expressed as a 46 kDa light chain level) in human plasma. The method includes pellet digestion after homogenizing 25 μL of plasma, further purification using MCX SPE, and analysis by liquid chromatography-tandem mass spectrometry (LC-MS / MS). Two-dimensional HPLC (Agilent Metasil AQ C18 column, 5.0 μm, 2.1 mm x 100 mm (C / N A0530100X020)) and an AB Sciex QTrap 6500 mass spectrometer were operated under optimized conditions in selected reaction monitoring (SRM) mode to detect the tagged peptide H2N-KHNLGHGH-OH (SEQ ID NO:1) of 2-chain HMWK using cations formed by electrospray ionization and a stable labeled internal standard (SLIS) (KHNL... 13 C6]GHGH).

[0256] An aliquot of 25 μL human plasma was mixed with 2.5 μL of 10% SDS, 5 μL of DTT (500 mM), and protein and reduced at 37 °C for 60 min. 75 μL and 600 μL of methanol were added sequentially to the solution to precipitate the protein. The supernatant was discarded, and the protein particles were washed with 700 μL of methanol. After centrifugation, the particles were reconstituted in 500 μL of ammonium bicarbonate buffer (100 mM) and vortexed vigorously for 10 min. 25 μL of internal standard (KHNL) was added. 13 An aliquot of [C6]GHGH (30 ng / mL) and 5.0 μL of iodoacetamide solution (500 mM) were added and the mixture was kept in the dark for 30 minutes. The protein was then digested by adding 10 μL of chymotrypsin (8 mg / mL) and incubating at 50°C for 3 hours with gentle vortexing.

[0257] The resulting mixture was acidified and packed into an MCX 96-well extraction column (30 μm, 10 mg) for desalting. The extraction column was washed with 900 μL of 2.0% formic acid, 900 μL of water, and 900 μL of methanol, respectively, and the resulting peptide was eluted with 600 μL of ammonium hydroxide in 5.0% methanol. The eluted solution was dried under nitrogen and reconstituted in 100 μL of methanol:water (10:90; v / v) containing 1.0% HFBA.

[0258] For sample analysis, the peptide was loaded into an LC-MS / MS instrument operating in MRM mode, where the tagged peptide KHNLGHGH (SEQ ID NO:1) and its internal standard KHNL were recorded. 13The ionic strength of C6]GHGH was determined. The relative content of the 46 kDa light chain of 2-chain HMWK in the plasma sample was calculated accordingly.

[0259] (i) Measurement and verification (precision and accuracy):

[0260] The methods described herein were used to determine the precision (%RSD) and accuracy (%RE) for intra-day (n=6) and inter-day (n=18) assays of the 46 kDa light chain in human plasma (SCAT169). As shown in Table 7 below, the levels of 46 kDa determined using the methods described herein in both intra-day and inter-day assays are very close to the theoretical concentration of 46 kDa in the test samples. These results demonstrate the accuracy of the assay methods described herein, using the tagged peptide of the 46 kDa light chain described above as a biomarker.

[0261] Table 7: Precision (%RSD) and accuracy (%RE) for intraday (n=6) and interday (n=18) quantification of 46 kDa light chains in human plasma (SCAT169)

[0262]

[0263] (ii) Stability assessment

[0264] Human plasma samples were collected in SCAT169 tubes as described herein. Samples underwent four freeze-thaw cycles or were held on the bench (4°C) for 8 hours. The assay described herein (using a 46kDa light chain-tagged peptide) was performed to measure the 46kDa light chain level in both samples. The results thus obtained indicate that 2-chain HMWK (represented by the 46kDa light chain) is stable in human plasma samples. Table 8.

[0265] Table 8: Stability test of 2-chain HMWK in human plasma (SCAT169) (n=3)

[0266]

[0267] (iii) Application of assay methods in disease diagnosis and prognosis

[0268] Plasma samples from healthy human subjects and HAE patients were collected into SCAT169 tubes. The assay described above was performed to measure the level of 2-chain HMWK in these human plasma samples. Figure 7 As shown, the 2-chain HMWK level in HAE patients was significantly higher than that in healthy human subjects. These results suggest that 2-chain HMWK and any of its components (such as the 46 kDa light chain) can be used as reliable biomarkers for the diagnosis and / or prognosis of HAE.

[0269] Other implementation plans

[0270] All features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification can be used to replace alternative features for the same, equivalent, or similar purposes. Therefore, unless otherwise expressly stated, each disclosed feature is merely one example of a general series of equivalent or similar features.

[0271] Based on the above description, those skilled in the art can readily determine the essential features of this disclosure, and various changes and modifications can be made to the invention to adapt it to various uses and conditions without departing from its spirit and scope. Therefore, other embodiments are also within the scope of the claims.

[0272] Equivalent content

[0273] Although several inventive embodiments have been described and illustrated herein, those skilled in the art will readily conceive of a variety of other devices and / or structures to perform the functions and / or obtain the results and / or one or more advantages described herein, and each of such changes and / or variations is considered to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials, and constructions described herein are exemplary, and actual parameters, dimensions, materials, and / or constructions will depend on one or more specific applications using the teachings of this invention. Those skilled in the art will recognize, or can determine, many equivalents to the specific inventive embodiments described herein using only conventional experimentation. Therefore, it is understood that the foregoing embodiments are presented merely as examples, and inventive embodiments may be practiced beyond those specifically described and claimed within the scope of the appended claims and their equivalents. The inventive embodiments of this disclosure are intended for each individual feature, system, article of manufacture, material, kit, and / or method described herein. Furthermore, if such features, systems, articles, materials, kits, and / or methods are not contradictory, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the scope of the inventive step of this disclosure.

[0274] All definitions used and defined herein should be understood to take precedence over dictionary definitions, definitions incorporated by reference in other documents, and / or their usual meaning.

[0275] Unless otherwise expressly stated, the indefinite articles “a” and “an” used in the specification and claims shall be understood to mean “at least one”.

[0276] The expression “and / or” as used in this specification and claims should be understood to mean “any one or both” of the elements thus combined, i.e., elements that exist jointly in some cases and separately in others. Multiple elements listed with “and / or” should be understood in the same way, i.e., “one or more” of the elements thus combined. Unlike elements explicitly indicated by a “and / or” clause, other elements may optionally be present, whether related to or unrelated to the explicitly indicated elements. Thus, as a non-limiting example, reference to “A and / or B”, when used with open-ended language such as “comprising,” can in one embodiment refer to only A (optionally including elements other than B); in another embodiment refer to only B (optionally including elements other than A); in yet another embodiment refer to both A and B (optionally including other elements), and so on.

[0277] As used herein in the specification and claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when items are listed separately, “or” or “and / or” should be understood to include, i.e., include at least one of a plurality of elements or a list of elements, and include more than one, as well as optional additional unlisted items. Only terms that express the opposite meaning, such as “only one” or “exact one” or when used in a claim, “consisting of…”, will refer to exactly one of a plurality of elements or a list of elements. Generally, when preceded by an exclusive term such as “one of both,” “one of,” “only one” or “exact one,” the term “or” as used herein should be interpreted to indicate an exclusive alternative (i.e., “one or the other, but not both”). “Substantially consisting of…” when used in a claim should have its usual meaning as used in the field of patent law.

[0278] Regarding the list of one or more elements, the expression "at least one" as used in this specification and claims should be understood to mean at least one element selected from any one or more elements in the list of elements, but does not necessarily include at least one of each element explicitly listed in the list of elements, nor exclude any combination of elements in the list of elements. This definition also allows for the optional presence of other elements besides those specifically identified in the list of elements referred to by the word "at least one," regardless of their relation to or irrelevance to those specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B," or equivalently, "at least one of A and / or B") in one embodiment may mean at least one A, optionally including more than one A, but without B (and optionally including elements other than B); in another embodiment, it may mean at least one B, optionally including more than one B, but without A (and optionally including elements other than A); in yet another embodiment, it may mean at least one A, optionally including more than one A, and at least one B, optionally including more than one B (and optionally including other elements), and so on.

[0279] It should also be understood that, unless otherwise stated to the contrary, in any method claimed herein that includes more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order of the steps or actions of the enumerated methods.

[0280] In the claims and the description above, all conjunctions such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “keeping,” “composed of,” and similar words may be understood as open-ended, for example, meaning including but not limited to. Only the conjunctions “consisting of” and “consisting essentially of” should be closed or semi-closed conjunctions, respectively, as set forth in section 2111.03 of the U.S. Patent Examination Manual. sequence list <110> Takeda Pharmaceutical Co., Ltd. <120> A peptide quantification method to differentiate between full-length high molecular weight kininogen (HMWK) and cleaved HMWK. <130> D0617.70114WO00 <140> 2016800798659 <141> 2016-12-15 <150> US 62 / 267,734 <151> 2015-12-15 <160> 30 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 1 Lys His Asn Leu Gly His Gly His 1 5 <210> 2 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 2 Lys His Asn Leu Gly His Gly His Lys His Glu 1 5 10 <210> 3 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 3 Lys His Asn Leu Gly His Gly His Lys 1 5 <210> 4 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 4 Lys His Asn Leu Gly His Gly His Lys His Glu Arg 1 5 10 <210> 5 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 5 Ser Ser Arg Ile Gly Glu 1 5 <210> 6 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 6 Gly His Glu Lys Gln Arg Lys His 1 5 <210> 7 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 7 Lys Gln Arg Lys His Asn Leu Gly His Gly His Lys His Glu 1 5 10 <210> 8 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 8 Asp Trp Gly His Lys Gln Arg Lys His Asn Leu Gly His Gly His Lys 1 5 10 15 His Glu Arg <210> 9 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 9 His Asn Leu Gly His Gly His Lys 1 5 <210> 10 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 10 Ser Tyr Tyr Phe Asp Leu Thr Asp Gly Leu Ser 1 5 10 <210> 11 <211> 644 <212> PRT <213> Homo sapiens <400> 11 Met Lys Leu Ile Thr Ile Leu Phe Leu Cys Ser Arg Leu Leu Leu Ser 1 5 10 15 Leu Thr Gln Glu Ser Gln Ser Glu Glu Ile Asp Cys Asn Asp Lys Asp 20 25 30 Leu Phe Lys Ala Val Asp Ala Ala Leu Lys Lys Tyr Asn Ser Gln Asn 35 40 45 Gln Ser Asn Asn Gln Phe Val Leu Tyr Arg Ile Thr Glu Ala Thr Lys 50 55 60 Thr Val Gly Ser Asp Thr Phe Tyr Ser Phe Lys Tyr Glu Ile Lys Glu 65 70 75 80 Gly Asp Cys Pro Val Gln Ser Gly Lys Thr Trp Gln Asp Cys Glu Tyr 85 90 95 Lys Asp Ala Ala Lys Ala Ala Thr Gly Glu Cys Thr Ala Thr Val Gly 100 105 110 Lys Arg Ser Ser Thr Lys Phe Ser Val Ala Thr Gln Thr Cys Gln Ile 115 120 125 Thr Pro Ala Glu Gly Pro Val Val Thr Ala Gln Tyr Asp Cys Leu Gly 130 135 140 Cys Val His Pro Ile Ser Thr Gln Ser Pro Asp Leu Glu Pro Ile Leu 145 150 155 160 Arg His Gly Ile Gln Tyr Phe Asn Asn Asn Thr Gln His Ser Ser Leu 165 170 175 Phe Met Leu Asn Glu Val Lys Arg Ala Gln Arg Gln Val Val Ala Gly 180 185 190 Leu Asn Phe Arg Ile Thr Tyr Ser Ile Val Gln Thr Asn Cys Ser Lys 195 200 205 Glu Asn Phe Leu Phe Leu Thr Pro Asp Cys Lys Ser Leu Trp Asn Gly 210 215 220 Asp Thr Gly Glu Cys Thr Asp Asn Ala Tyr Ile Asp Ile Gln Leu Arg 225 230 235 240 Ile Ala Ser Phe Ser Gln Asn Cys Asp Ile Tyr Pro Gly Lys Asp Phe 245 250 255 Val Gln Pro Pro Thr Lys Ile Cys Val Gly Cys Pro Arg Asp Ile Pro 260 265 270 Thr Asn Ser Pro Glu Leu Glu Glu Thr Leu Thr His Thr Ile Thr Lys 275 280 285 Leu Asn Ala Glu Asn Asn Ala Thr Phe Tyr Phe Lys Ile Asp Asn Val 290 295 300 Lys Lys Ala Arg Val Gln Val Val Ala Gly Lys Lys Tyr Phe Ile Asp 305 310 315 320 Phe Val Ala Arg Glu Thr Thr Cys Ser Lys Glu Ser Asn Glu Glu Leu 325 330 335 Thr Glu Ser Cys Glu Thr Lys Lys Leu Gly Gln Ser Leu Asp Cys Asn 340 345 350 Ala Glu Val Tyr Val Val Pro Trp Glu Lys Lys Ile Tyr Pro Thr Val 355 360 365 Asn Cys Gln Pro Leu Gly Met Ile Ser Leu Met Lys Arg Pro Pro Gly 370 375 380 Phe Ser Pro Phe Arg Ser Ser Arg Ile Gly Glu Ile Lys Glu Glu Thr 385 390 395 400 Thr Val Ser Pro Pro His Thr Ser Met Ala Pro Ala Gln Asp Glu Glu 405 410 415 Arg Asp Ser Gly Lys Glu Gln Gly His Thr Arg Arg His Asp Trp Gly 420 425 430 His Glu Lys Gln Arg Lys His Asn Leu Gly His Gly His Lys His Glu 435 440 445 Arg Asp Gln Gly His Gly His Gln Arg Gly His Gly Leu Gly His Gly 450 455 460 His Glu Gln Gln His Gly Leu Gly His Gly His Lys Phe Lys Leu Asp 465 470 475 480 Asp Asp Leu Glu His Gln Gly Gly His Val Leu Asp His Gly His Lys 485 490 495 His Lys His Gly His Gly His Gly Lys His Lys Asn Lys Gly Lys Lys 500 505 510 Asn Gly Lys His Asn Gly Trp Lys Thr Glu His Leu Ala Ser Ser Ser 515 520 525 Glu Asp Ser Thr Thr Pro Ser Ala Gln Thr Gln Glu Lys Thr Glu Gly 530 535 540 Pro Thr Pro Ile Pro Ser Leu Ala Lys Pro Gly Val Thr Val Thr Phe 545 550 555 560 Ser Asp Phe Gln Asp Ser Asp Leu Ile Ala Thr Met Met Pro Pro Ile 565 570 575 Ser Pro Ala Pro Ile Gln Ser Asp Asp Asp Trp Ile Pro Asp Ile Gln 580 585 590 Ile Asp Pro Asn Gly Leu Ser Phe Asn Pro Ile Ser Asp Phe Pro Asp 595 600 605 Thr Thr Ser Pro Lys Cys Pro Gly Arg Pro Trp Lys Ser Val Ser Glu 610 615 620 Ile Asn Pro Thr Thr Gln Met Lys Glu Ser Tyr Tyr Phe Asp Leu Thr 625 630 635 640 Asp Gly Leu Ser <210> 12 <211> 427 <212> PRT <213> Homo sapiens <400> 12 Met Lys Leu Ile Thr Ile Leu Phe Leu Cys Ser Arg Leu Leu Leu Ser 1 5 10 15 Leu Thr Gln Glu Ser Gln Ser Glu Glu Ile Asp Cys Asn Asp Lys Asp 20 25 30 Leu Phe Lys Ala Val Asp Ala Ala Leu Lys Lys Tyr Asn Ser Gln Asn 35 40 45 Gln Ser Asn Asn Gln Phe Val Leu Tyr Arg Ile Thr Glu Ala Thr Lys 50 55 60 Thr Val Gly Ser Asp Thr Phe Tyr Ser Phe Lys Tyr Glu Ile Lys Glu 65 70 75 80 Gly Asp Cys Pro Val Gln Ser Gly Lys Thr Trp Gln Asp Cys Glu Tyr 85 90 95 Lys Asp Ala Ala Lys Ala Ala Thr Gly Glu Cys Thr Ala Thr Val Gly 100 105 110 Lys Arg Ser Ser Thr Lys Phe Ser Val Ala Thr Gln Thr Cys Gln Ile 115 120 125 Thr Pro Ala Glu Gly Pro Val Val Thr Ala Gln Tyr Asp Cys Leu Gly 130 135 140 Cys Val His Pro Ile Ser Thr Gln Ser Pro Asp Leu Glu Pro Ile Leu 145 150 155 160 Arg His Gly Ile Gln Tyr Phe Asn Asn Asn Thr Gln His Ser Ser Leu 165 170 175 Phe Met Leu Asn Glu Val Lys Arg Ala Gln Arg Gln Val Val Ala Gly 180 185 190 Leu Asn Phe Arg Ile Thr Tyr Ser Ile Val Gln Thr Asn Cys Ser Lys 195 200 205 Glu Asn Phe Leu Phe Leu Thr Pro Asp Cys Lys Ser Leu Trp Asn Gly 210 215 220 Asp Thr Gly Glu Cys Thr Asp Asn Ala Tyr Ile Asp Ile Gln Leu Arg 225 230 235 240 Ile Ala Ser Phe Ser Gln Asn Cys Asp Ile Tyr Pro Gly Lys Asp Phe 245 250 255 Val Gln Pro Pro Thr Lys Ile Cys Val Gly Cys Pro Arg Asp Ile Pro 260 265 270 Thr Asn Ser Pro Glu Leu Glu Glu Thr Leu Thr His Thr Ile Thr Lys 275 280 285 Leu Asn Ala Glu Asn Asn Ala Thr Phe Tyr Phe Lys Ile Asp Asn Val 290 295 300 Lys Lys Ala Arg Val Gln Val Val Ala Gly Lys Lys Tyr Phe Ile Asp 305 310 315 320 Phe Val Ala Arg Glu Thr Thr Cys Ser Lys Glu Ser Asn Glu Glu Leu 325 330 335 Thr Glu Ser Cys Glu Thr Lys Lys Leu Gly Gln Ser Leu Asp Cys Asn 340 345 350 Ala Glu Val Tyr Val Val Pro Trp Glu Lys Lys Ile Tyr Pro Thr Val 355 360 365 Asn Cys Gln Pro Leu Gly Met Ile Ser Leu Met Lys Arg Pro Pro Gly 370 375 380 Phe Ser Pro Phe Arg Ser Ser Arg Ile Gly Glu Ile Lys Glu Glu Thr 385 390 395 400 Thr Ser His Leu Arg Ser Cys Glu Tyr Lys Gly Arg Pro Pro Lys Ala 405 410 415 Gly Ala Glu Pro Ala Ser Glu Arg Glu Val Ser 420 425 <210> 13 <211> 362 <212> PRT <213> Homo sapiens <400> 13 Gln Glu Ser Gln Ser Glu Glu Ile Asp Cys Asn Asp Lys Asp Leu Phe 1 5 10 15 Lys Ala Val Asp Ala Ala Leu Lys Lys Tyr Asn Ser Gln Asn Gln Ser 20 25 30 Asn Asn Gln Phe Val Leu Tyr Arg Ile Thr Glu Ala Thr Lys Thr Val 35 40 45 Gly Ser Asp Thr Phe Tyr Ser Phe Lys Tyr Glu Ile Lys Glu Gly Asp 50 55 60 Cys Pro Val Gln Ser Gly Lys Thr Trp Gln Asp Cys Glu Tyr Lys Asp 65 70 75 80 Ala Ala Lys Ala Ala Thr Gly Glu Cys Thr Ala Thr Val Gly Lys Arg 85 90 95 Ser Ser Thr Lys Phe Ser Val Ala Thr Gln Thr Cys Gln Ile Thr Pro 100 105 110 Ala Glu Gly Pro Val Val Thr Ala Gln Tyr Asp Cys Leu Gly Cys Val 115 120 125 His Pro Ile Ser Thr Gln Ser Pro Asp Leu Glu Pro Ile Leu Arg His 130 135 140 Gly Ile Gln Tyr Phe Asn Asn Asn Thr Gln His Ser Ser Leu Phe Met 145 150 155 160 Leu Asn Glu Val Lys Arg Ala Gln Arg Gln Val Val Ala Gly Leu Asn 165 170 175 Phe Arg Ile Thr Tyr Ser Ile Val Gln Thr Asn Cys Ser Lys Glu Asn 180 185 190 Phe Leu Phe Leu Thr Pro Asp Cys Lys Ser Leu Trp Asn Gly Asp Thr 195 200 205 Gly Glu Cys Thr Asp Asn Ala Tyr Ile Asp Ile Gln Leu Arg Ile Ala 210 215 220 Ser Phe Ser Gln Asn Cys Asp Ile Tyr Pro Gly Lys Asp Phe Val Gln 225 230 235 240 Pro Pro Thr Lys Ile Cys Val Gly Cys Pro Arg Asp Ile Pro Thr Asn 245 250 255 Ser Pro Glu Leu Glu Glu Thr Leu Thr His Thr Ile Thr Lys Leu Asn 260 265 270 Ala Glu Asn Asn Ala Thr Phe Tyr Phe Lys Ile Asp Asn Val Lys Lys 275 280 285 Ala Arg Val Gln Val Val Ala Gly Lys Lys Tyr Phe Ile Asp Phe Val 290 295 300 Ala Arg Glu Thr Thr Cys Ser Lys Glu Ser Asn Glu Glu Leu Thr Glu 305 310 315 320 Ser Cys Glu Thr Lys Lys Leu Gly Gln Ser Leu Asp Cys Asn Ala Glu 325 330 335 Val Tyr Val Val Pro Trp Glu Lys Lys Ile Tyr Pro Thr Val Asn Cys 340 345 350 Gln Pro Leu Gly Met Ile Ser Leu Met Lys 355 360 <210> 14 <211> 255 <212> PRT <213> Homo sapiens <400> 14 Ser Ser Arg Ile Gly Glu Ile Lys Glu Glu Thr Thr Val Ser Pro Pro 1 5 10 15 His Thr Ser Met Ala Pro Ala Gln Asp Glu Glu Arg Asp Ser Gly Lys 20 25 30 Glu Gln Gly His Thr Arg Arg His Asp Trp Gly His Glu Lys Gln Arg 35 40 45 Lys His Asn Leu Gly His Gly His Lys His Glu Arg Asp Gln Gly His 50 55 60 Gly His Gln Arg Gly His Gly Leu Gly His Gly His Glu Gln Gln His 65 70 75 80 Gly Leu Gly His Gly His Lys Phe Lys Leu Asp Asp Asp Leu Glu His 85 90 95 Gln Gly Gly His Val Leu Asp His Gly His Lys His Lys His Gly His 100 105 110 Gly His Gly Lys His Lys Asn Lys Gly Lys Lys Asn Gly Lys His Asn 115 120 125 Gly Trp Lys Thr Glu His Leu Ala Ser Ser Ser Glu Asp Ser Thr Thr 130 135 140 Pro Ser Ala Gln Thr Gln Glu Lys Thr Glu Gly Pro Thr Pro Ile Pro 145 150 155 160 Ser Leu Ala Lys Pro Gly Val Thr Val Thr Phe Ser Asp Phe Gln Asp 165 170 175 Ser Asp Leu Ile Ala Thr Met Met Pro Pro Ile Ser Pro Ala Pro Ile 180 185 190 Gln Ser Asp Asp Asp Trp Ile Pro Asp Ile Gln Ile Asp Pro Asn Gly 195 200 205 Leu Ser Phe Asn Pro Ile Ser Asp Phe Pro Asp Thr Thr Ser Pro Lys 210 215 220 Cys Pro Gly Arg Pro Trp Lys Ser Val Ser Glu Ile Asn Pro Thr Thr 225 230 235 240 Gln Met Lys Glu Ser Tyr Tyr Phe Asp Leu Thr Asp Gly Leu Ser 245 250 255 <210> 15 <211> 207 <212> PRT <213> Homo sapiens <400> 15 Lys His Asn Leu Gly His Gly His Lys His Glu Arg Asp Gln Gly His 1 5 10 15 Gly His Gln Arg Gly His Gly Leu Gly His Gly His Glu Gln Gln His 20 25 30 Gly Leu Gly His Gly His Lys Phe Lys Leu Asp Asp Asp Leu Glu His 35 40 45 Gln Gly Gly His Val Leu Asp His Gly His Lys His Lys His Gly His 50 55 60 Gly His Gly Lys His Lys Asn Lys Gly Lys Lys Asn Gly Lys His Asn 65 70 75 80 Gly Trp Lys Thr Glu His Leu Ala Ser Ser Ser Glu Asp Ser Thr Thr 85 90 95 Pro Ser Ala Gln Thr Gln Glu Lys Thr Glu Gly Pro Thr Pro Ile Pro 100 105 110 Ser Leu Ala Lys Pro Gly Val Thr Val Thr Phe Ser Asp Phe Gln Asp 115 120 125 Ser Asp Leu Ile Ala Thr Met Met Pro Pro Ile Ser Pro Ala Pro Ile 130 135 140 Gln Ser Asp Asp Asp Trp Ile Pro Asp Ile Gln Ile Asp Pro Asn Gly 145 150 155 160 Leu Ser Phe Asn Pro Ile Ser Asp Phe Pro Asp Thr Thr Ser Pro Lys 165 170 175 Cys Pro Gly Arg Pro Trp Lys Ser Val Ser Glu Ile Asn Pro Thr Thr 180 185 190 Gln Met Lys Glu Ser Tyr Tyr Phe Asp Leu Thr Asp Gly Leu Ser 195 200 205 <210> 16 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 16 Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala 1 5 10 15 Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30 Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 <210> 17 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 17 Asp Trp Gly His Glu Lys Gln Arg Lys His Asn Leu Gly His Gly His 1 5 10 15 Lys His Glu Arg 20 <210> 18 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 18 Lys Lys Ile Tyr Pro Thr Val Asn Cys Gln Pro Leu Gly Met Ile Ser 1 5 10 15 Leu Met Lys Arg Pro Pro Gly Phe Ser Pro Phe Arg Ser Ser Arg Ile 20 25 30 Gly Glu <210> 19 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 19 Ser Ser Arg Ile Gly Glu Ile Lys Glu 1 5 <210> 20 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 20 Lys Lys Ile Tyr Pro Thr Val Asn Cys Gln Pro Leu Gly Met Ile Ser 1 5 10 15 Leu Met Lys Arg Pro Pro Gly Phe Ser Pro Phe Arg Ser Ser Arg Ile 20 25 30 Gly Glu <210> twenty one <211> 19 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> twenty one Lys Lys Ile Tyr Pro Thr Val Asn Cys Gln Pro Leu Gly Met Ile Ser 1 5 10 15 Leu Met Lys <210> twenty two <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> twenty two Ser Ser Arg Arg Ile Gly Glu 1 5 <210> twenty three <211> 19 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> twenty three Lys Lys Tyr Ile Pro Thr Val Asn Cys Gln Pro Leu Gly Met Ile Ser 1 5 10 15 Leu Met Lys <210> twenty four <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> twenty four Arg Pro Pro Gly Phe Ser Pro Phe Arg 1 5 <210> 25 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 25 Ser Ser Arg Ile Gly Glu Lys Glu 1 5 <210> 26 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 26 Ile Asn Pro Thr Thr Gln Met Lys Glu 1 5 <210> 27 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 27 Glu Asp Ser Thr Thr Pro Ser Ala Gln Thr Gln Glu 1 5 10 <210> 28 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 28 Tyr Lys Gly Arg Pro Pro Lys Ala Gly Ala Glu 1 5 10 <210> 29 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 29 Ile Asn Pro Thr Gln Met Lys Glu 1 5 <210> 30 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 30 Ser Tyr Tyr Asp Asp Gly Leu Ser 1 5

Claims

1. Use of a tag peptide-specific detection reagent in a kit for manufacturing a method for detecting cleaved high molecular weight kininogen (HMWK) in a sample, the detection comprising: (i) Provide samples suspected of containing HMWK; (ii) Contact the sample with a protease to produce a variety of digestible peptides; and (iii) The level of the tagged peptide in the plurality of digested peptides is measured by the detection reagent, wherein the tagged peptide indicates a 46 kDa light chain or a 56 kDa light chain of cleaved HMWK, wherein the tagged peptide indicating a 46 kDa light chain is KHNLGHGH (SEQ ID NO: 1), or wherein the tagged peptide indicating a 56 kDa light chain is SSRIGE (SEQ ID NO: 5).

2. The use as claimed in claim 1, wherein the protease is selected from the group consisting of: chymotrypsin, intracellular protease Glu-C, and cathepsin G.

3. The use as described in claim 1, further comprising measuring the level of the tagged peptide indicating full-length HMWK.

4. The use as claimed in claim 3, wherein the tag peptide indicating the full-length HMWK is SYYFDLTDGLS (SEQ ID NO: 10).

5. The use as described in any one of claims 1-4, wherein the level of the tag peptide indicating cleaved HMWK, the level of the tag peptide indicating full-length HMWK, or both the level of the tag peptide indicating cleaved HMWK and the level of the tag peptide indicating full-length HMWK are measured by liquid chromatography-mass spectrometry (LC-MS).

6. The use as claimed in claim 1, wherein the sample is a biological sample obtained from a human subject.

7. The use as described in claim 6, wherein the biological sample is a blood sample or a plasma sample.

8. The use as claimed in claim 7, wherein the plasma sample is a normal plasma sample, a plasma sample activated by FXIIa, or an unactivated plasma sample.

9. The use as claimed in claim 6, wherein the human subject has hereditary angioedema (HAE) or is suspected of having hereditary angioedema (HAE).

10. The use as claimed in claim 6, wherein the biological sample is a plasma sample collected in a vacuum-sealed blood collection tube, the vacuum-sealed blood collection tube containing a liquid formulation comprising a mixture of protease inhibitors.

11. The use as claimed in claim 10, wherein the vacuum-evacuated blood collection tube is an SCAT tube.

12. The use as claimed in claim 1, wherein step (ii) is performed in the presence of a reducing agent.

13. The use as claimed in claim 12, wherein the sample is incubated with the reducing agent at 90°C for 1 hour.

14. The use as claimed in claim 1, wherein step (ii) is performed in the absence of a protease inhibitor, an anticoagulant, or both a protease inhibitor and an anticoagulant.

15. The use of a first detection reagent specific to a first digestible peptide and a second detection reagent specific to a second digestible peptide in the manufacture of a kit for a method of distinguishing between full-length high molecular weight kininogen (HMWK) and cleaved HMWK in a sample, the distinction comprising: (i) Provide a sample suspected of containing full-length HMWK and / or cleaved HMWK; (ii) Contact the sample with a protease to produce a variety of digestible peptides; (iii) Measure the level of the first digestible peptide obtained from step (ii) using the first detection reagent, wherein the first digestible peptide is specific to the cleaved HMWK compared to the full-length HMWK; (iv) Measure the level of the second digestive peptide obtained from step (ii) using the second detection reagent, wherein the second digestive peptide is HMWK-specific compared to low molecular weight kininogen (LMWK); (v) Determine the ratio between the first digestible peptide and the second digestible peptide; and (vi) Based on the ratio determined in step (v), distinguish between the fragmented HMWK and the full-length HMWK. The first digestible peptide is SSRIGE (SEQ ID NO: 5) and the second digestible peptide is SYYFDLTDGLS (SEQ ID NO: 10).

16. The use as claimed in claim 15, wherein the protease is cleaved after a glutamate residue.

17. The use as described in claim 16, wherein the protease is an intracellular protease Glu-C or cathepsin G.

18. The use as claimed in claim 17, wherein the protease is an intracellular protease Glu-C.

19. The use of any one of claims 15-18, wherein the first digestible peptide and the second digestible peptide are measured by liquid chromatography-mass spectrometry (LC-MS).

20. The use as claimed in claim 15, wherein the sample is a biological sample obtained from a human subject.

21. The use as described in claim 20, wherein the biological sample is a blood sample or a plasma sample.

22. The use as claimed in claim 21, wherein the plasma sample is a normal plasma sample, a plasma sample activated by FXIIa, or an unactivated plasma sample.

23. The use as claimed in claim 20, wherein the human subject has hereditary angioedema (HAE) or is suspected of having hereditary angioedema (HAE).

24. The use as claimed in claim 20, wherein the biological sample is a plasma sample collected in a vacuum-sealed blood collection tube, the vacuum-sealed blood collection tube containing a liquid formulation comprising a mixture of protease inhibitors.

25. The use as described in claim 24, wherein the vacuum-evacuated blood collection tube is an SCAT tube.

26. The use as claimed in claim 15, wherein step (ii) is performed in the presence of a reducing agent.

27. The use as claimed in claim 26, wherein the sample is incubated with the reducing agent at 90°C for 1 hour.

28. The use as claimed in claim 15, wherein step (ii) is performed in the absence of a protease inhibitor, an anticoagulant, or both a protease inhibitor and an anticoagulant.

29. The use as claimed in claim 15, wherein the protease / protein ratio in step (ii) is 1:

20.

30. Use of a first assay reagent specific to a first tag peptide in a kit for manufacturing a method for detecting cleaved high molecular weight kininogen (HMWK) in subjects suffering from or at risk of hereditary angioedema (HAE), said assay comprising: (i) Provide biological samples from the subjects; (ii) Contact the sample with a protease to produce a variety of digestible peptides; and (iii) Measure the level of the first-tagged peptide among the plurality of digestible peptides by means of the first-tagged peptide, wherein the first-tagged peptide indicates cleaved HMWK; The first tag peptide is KHNLGHGH (SEQ ID NO: 1) or SSRIGE (SEQ ID NO: 5).

31. The use as described in claim 30, wherein the protease is chymotrypsin, intracellular protease Glu-C, or cathepsin.

32. The use as claimed in claim 30, further comprising a second detection reagent specific to the second tagged peptide, wherein the detection further comprises measuring the level of the second tagged peptide among the plurality of digestible peptides by the second detection reagent, and the second tagged peptide indicating HMWK.

33. The use as described in claim 32, further comprising determining the ratio between the first tagged peptide and the second tagged peptide.

34. The use as described in claim 32, wherein the second tag peptide is SYYFDLTDGLS (SEQ ID NO: 10).

35. The use as described in any one of claims 30-34, wherein the first tagged peptide, the second tagged peptide, or both the first tagged peptide and the second tagged peptide are measured by LC-MS.

36. The use as described in claim 30, wherein the biological sample is a blood sample or a plasma sample.

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