Methods for detecting protein cleavage

Abstract

A method for detecting cleavage of a target protein or protein of interest (POI) in a non-purified sample, wherein the target protein comprises a first signature peptide or domain and a second signatu

Description

The present invention relates to methods for determining whether a protein within or on a cell in a non-purified sample has been cleaved, and to methods for detecting apoptosis in a cell in a non-purified sample. Thermal shift assays have been developed in the art which can assess protein-ligand binding where the protein is in purified form. These assays have been developed on the basis of two principles, namely that a purified protein will melt and unfold at a particular temperature and that the binding of a ligand to a protein will thermally stabilise the protein. Thus, the binding of a ligand to a protein can be detected on the basis that the purified protein will show an increase in thermal stability once a ligand is bound and hence the protein will melt at a higher temperature once ligand is bound than purified protein alone. The methods of the invention comprise the use of Cellular Thermal Shift Assay (CETSA), which is described in WO 2012 / 143714A1. Prior to the invention of CETSA, thermal shift assays known in the field were only used in connection with purified protein. WO2012 / 143714 describes the use of CETSA for detecting stability changes of target proteins within or on intact cells following drug-target protein binding, in non-purified samples, i.e. without purification of the target protein. CETSA allows for global studies of protein stability changes in intact cells, primarily reflecting interaction changes for proteins. Most applications of CETSA have so far focused on identifying the direct binding of drugs with their cognate protein target(s), including the use of proteome-wide mass-spectrometry CETSA (MS-CETSA) for discovering new target proteins. At the same time, multiple explorative studies have established that CETSA can yield distinct information on the interaction changes between proteins with other cellular molecules. However, for a CETSA shift to be measurable, interaction / biochemical changes are required at relatively high cellular stoichiometries, with protein stability significantly affected by the interaction(s). This presents challenges and an expected limitation on the utility of the method in analyzing less dominant intracellular effects, particularly in complex, non-purified samples. To date, CETSA tools and methods are predicated on protein stabilization / de-stabilization at the protein level. Further refined methods of analyzing stability changes of proteins within or on intact cells in non-purified samples would be desirable. In particular, it would be desirable to be able to detect changes in a protein’s structural state beyond stabilization / de-stabilization of the complete protein. However, given that CETSA assays are performed using non-purified (i.e. complex) samples, there are low expectations regarding the potential for the method to yield more detailed analysis of protein stabilization. The present inventors have however unexpectedly identified such a refined and improved CETSA method, which permits detection of cleavage of a protein of interest in a complex sample. Apoptosis is a repertoire of controlled cellular processes that eventually lead to cell death and systematic degradation of cellular components. Apoptosis is a form of programmed cell death in which cells are degraded in an ordered form. Apoptosis inducing pathway are typically divided into extrinsic and intrinsic pathways. The textbook scenario is that extrinsic pathways are induced by extracellular signals leading to intracellular activation of caspase 8, an initiator caspase, whereas apoptosis proceeding through the intrinsic pathway is typical induced by some type of cellular stress, leading to activation of initiator caspase 9. The initiator caspases subsequently cleave and activate effector caspases such as caspase 3 and 7. Apoptosis plays important roles in a range of pathological conditions including neurodegenerative, immune suppressant, liver and infection diseases. Apoptosis is also an important component in the action of some cytotoxic drugs such as anti-cancer or immune suppressant drugs. Thus, activation of cellular pathways that block apoptosis is a means for cancer cells to establish resistance against anti-cancer drugs. In neurological diseases such as Alzheimer’s or Parkinson diseases, the attenuation of apoptotic processes by drugs is desirable to slow disease progression. Therefore stringent methods to detect apoptosis are valuable as research tools and in clinical assays. Typically, the detection of apoptosis within a cell is performed with imaging or cell sorting techniques, which are time consuming and / or do not yield details about the apoptosis process and / or do not adequately detect apoptosis during the earliest stages of the apoptotic process. Examples of assays used to detect specific components of apoptosis include the annexin 5 assay in which distortions of lipid organisation on the cell membrane induced by apoptosis is detected, and assays detecting caspase activity or the cleavage of caspase targets. These assays are however not always applicable and can be prone to experimental errors. There remains a need in the field for further and improved methods of detecting apoptosis. Particularly advantageous would be methods for detecting apoptosis earlier in the apoptotic process than can be detected using imaging or sorting techniques. The present inventors have identified an advantageous method for detecting apoptosis, which advantageously permits detection soon after the commencement of apoptosis. In one aspect, the present invention provides a method for detecting cleavage of a protein of interest in a non-purified test sample, wherein said protein of interest comprises at least a first signature peptide present in a first domain and at least a second signature peptide present in a second domain, wherein the method comprises the steps of: a) heating the non-purified test sample to provide a heated test sample; b) separating soluble from insoluble protein in the heated test sample of step a) to produce soluble and insoluble protein fractions of said heated test sample; and c) determining, in the soluble and / or the insoluble protein fraction of step b), i) a first relative level, which is the level of the first signature peptide as compared to a control level, and ii) a second relative level, which is the level of the second signature peptide as compared to a control level, wherein cleavage is detected by a difference between said first and second relative levels in the soluble protein fraction and / or by a difference between said first and second relative levels in the insoluble protein fraction. In all aspects and embodiments described herein, reference to the level of a peptide is a reference to the level of a signature peptide. In all aspects and embodiments described herein, reference to determining or comparing a “level” of a signature peptide or group thereof may be the level in the soluble and / or the insoluble protein fraction, unless it is clearly stated or it is clear from the technical context that the level in the soluble protein fraction in particular is required, or the level in the soluble protein fraction in particular is required. The methods of the invention for detecting cleavage of a protein of interest can be alternatively viewed as methods for determining the cleavage status of the protein of interest. The methods of the invention detect cleavage or determine the cleavage status of a protein of interest in the non-purified test sample prior to the performance of the method, i.e. before the heating step of the method. In some embodiments, the steps of the methods of the invention themselves comprise digesting (i.e. cleaving) proteins in the sample after heating, or in an aliquot or protein fraction of a heated sample, to facilitate analysis, but the methods of the invention achieve detect cleavage or the determination of the cleavage status of the protein of interest in the non-purified sample prior to the heating step, and prior to such active digestion steps that may be performed as part of the method. A “protein of interest “ as used herein is any protein. The protein may be a prokaryotic or eukaryotic protein. Preferably the protein is a mammalian protein, more preferably a human protein. The protein may be a non-human animal protein, e.g. from a dog, cat, monkey, rabbit, mouse, rat, etc. The protein of interest may be a plant protein, or a bacterial protein. The protein of interest may be of known identity prior to the performance of the method of the invention. Alternatively, the protein of interest may be of unknown identity prior to the performance of the method of the invention. Thus, the present invention also provides a method for identifying cleaved proteins within a non-purified sample; the processed samples can be analysed by mass spectrometry or by using affinity binding moieties against a known target domain in order to identify the cleaved protein. It is straightforward to determine the sequence of peptides or domains detected by the present methods, and to identify them as components of a particular protein in the proteome of a particular organism by reference to sequence databases. Preferably, the protein of interest is within or on a cell, i.e. the method is performed on a non-purified test sample comprising a cell (or population of cells) that comprise the protein of interest. The cell may be any cell, including a prokaryotic or eukaryotic cell. Preferably the cell is a mammalian cell, more preferably a human cell e.g. a cell from a human patient. The cell may be an animal cell, particularly a cell from an animal patient, or a disease model, e.g. dog, cat, monkey, rabbit, mouse, rat, etc.. The cell may be a plant cell or bacterial cell. A protein “within” a cell is a protein located on the interior of the cell, i.e. inside the space defined by the cell membrane, for instance but not limited to in the cytosol or nucleus. A protein “on” a cell is a protein located accessible from outside the cell, i.e. from the exterior of the cell membrane, and may be tethered or otherwise anchored to the cell membrane. Proteins located in the cell membrane and on only the internal side of the cell membrane would be considered to be “within the cell”. Proteins spanning the cell membrane may be considered to be proteins both “within” and “on” the cell. However, these distinctions are not relevant to the present methods, which permit determination of the cleavage status of any protein that is within or on the cell. As discussed below, the method may be performed on a non-purified sample that does not comprise intact cells. Examples include cell extracts, lysates, urine and blood plasma. In such samples, the protein of interest is not within or on a cell, but the samples are still complex, and are non-purified samples. The methods of the invention permit detection of the cleavage of a protein of interest in such samples. Preferably, the protein of interest is not a target for a drug that is applied to the sample or administered to the subject from which the sample is obtained. Preferably in some aspects and embodiments, the protein of interest is selected from the group consisting of PARP1, DDX21, LMNB2, LMNB1, BCAP31 and MATR3. Cleavage of one or more of these proteins detected via a method of the present invention has been identified by the present inventors as a biomarker of apoptosis, particularly where apoptosis in which caspases are activated. The biomarker proteins named herein (detailed below) are human proteins, but the biomarker proteins may be homologous proteins from other species. Biomarker Protein Name Uniprot Accession No. BCAP31 B-cell receptor-associated protein 31 P51572 DDX21 ATP-dependent RNA helicase DDX21 Q9NR30 LMNB1 Lamin-B 1 P20700 LMNB2 Lamin-B2 Q03252 MATR3 Matrin-3 P43243 PARP1 Poly [ADP-ribose] polymerase 1 P09874 As used herein, detecting “cleavage” of a protein of interest means determining whether one or more covalent bonds within the primary amino acid sequence of the protein has been cleaved, resulting in two or more separate protein domains. The methods of the invention are not concerned with detecting breakage of disulphide bonds in higher order protein structures, since the cleavage of such bonds does not break the primary amino acid sequence of the peptide. Likewise, the methods of the invention are not concerned with detecting mere dissociation of higher order protein structures bound by non-covalent interactions, e.g. the binding of monomers within a dimeric, trimeric or multimeric protein structure. The protein of interest comprises at least a first and second protein domain. As used herein, a protein “domain” is a protein “region”, i.e. a continuous region of a protein. The person of ordinary skill in the art is aware that a structural domain is a region of a protein's polypeptide chain that is self-stabilizing and folds independently. Each structural domain forms a compact folded three-dimensional structure. A protein domain as the term is used herein, i.e. a protein region, may comprise one or more structural domains. A domain (i.e. region) as referred to herein may comprise or consist of a single structural domain, In all aspects and embodiments herein, the terms “domain” and “region” are interchangeable. The different domains for a given protein of interest are readily identifiable from the literature and publicly available protein databases, such as Interpro or Pfam. A domain is not defined by its size, but may comprise, for instance, 30 to 700 amino acids, e.g. 50 to 250 or 50 to 200 amino acids. The proteins of interest herein comprise at least a first domain and a second domain. The first and second domains are located on opposite sides (i.e. are on either side) of a cleavage site in the protein of interest. The cleavage site need not be a known cleavage site. However, the cleavage site may be a known cleavage site. The first and second signature peptides may be known to be present on opposite sides of a known cleavage site. The cleavage site is preferably a caspase cleavage site, and may be a known or unknown caspase cleavage site. Caspases cleave proteins in lysates at sequences which are typically enriched on glutamate, aspartate or small hydrophobic residues, but in the cell, caspase specificity is likely highly regulated by localisation and interactions with other proteins determining which substrate proteins can be accessed and cleaved The first and second domains have non-overlapping amino acid sequences i.e. the sequences of the first and second domains are each found within the amino acid sequence of the protein of interest, but the sequences are discrete sequences lacking any region of overlap. The domains may be separated by, for instance, at least 5, 10, 20, or 50 amino acids in the protein of interest. Preferably, the first domain is an N-terminal domain and the second domain is a C-terminal domain, or vice versa. Proteins comprise an amino acid sequence; this is a protein’s primary structure. Any linear portion of the protein may be referred to as a peptide, or a polypeptide, having a peptide (or polypeptide) sequence. The terms polypeptide and peptide are used interchangeably. Thus a protein (or a protein domain) can be thought of as comprising multiple peptides and a protein’s (or a protein domain’s) amino acid sequence can be thought of as comprising multiple peptide sequences. For instance amino acids 1-10 of the protein (or domain) may be considered one peptide, amino acids 2-11 may be considered as another peptide, amino acids 3-12 may be considered as another peptide, and so on. Peptides need not be 10 amino acids in length; they may be of any length. In the above example, amino acids 11-15 may be considered another peptide, as may amino acids 16-20, 8-15, 9-15, 9-16, 12-20, and so on. In other words, the protein (or domain) comprises a series of contiguous, optionally overlapping, peptide sequences. A “peptide” as referred to herein is derived from a protein (or a protein domain). The methods of the invention comprise the determination of levels of at least two peptides from a protein of interest, thus, by definition, the peptides referred to are not “full length” proteins. A protein can be considered to comprise “signature peptides”. As used herein, a “signature peptide” is a peptide (as defined above) comprising an amino acid sequence that is specific (i.e. unique) to a given protein. In other words, a signature peptide of a protein of interest comprises a sequence present in the protein of interest and only in the protein of interest, i.e. the signature peptide sequence is not found elsewhere in the proteome of the cell in which the protein of interest is found, preferably is not found elsewhere in the proteome of the sample. In the methods of the present invention, a signature peptide may be of any length provided that it is specific (i.e. unique) to the protein of interest. Typically, signature peptides are at least 5, preferably at least 10, 15 or 20 amino acids in length. Signature peptides may be, for instance, 5 to 25 amino acids in length, 6 to 20 amino acids in length, or 7 to 15 amino acids in length. However, the signature peptides may also be much larger peptides, e.g. at least 30, 40, 50, 75 or 100, 150 or 200 amino acids in length. There is no numerical upper limit to the length of a signature peptide, but as mentioned above, the methods of the invention comprise determining the levels of at least two signature peptides from the protein of interest. The first and second signature peptides are present in different domains of the protein of interest. The first and second signature peptides are thus non-overlapping i.e. the sequences of the first and second signature peptides are each found within the amino acid sequence of the protein of interest, but the sequences are discrete sequences lacking any region of overlap. They are located on opposite sides (i.e. are on either side) of a cleavage site. As mentioned above, he cleavage site need not be a known cleavage site. In practice, if different levels of two or more signature peptide sequences are determined, then this is indicative of the protein of interest having been cleaved, and the signature peptides being present in different domains that are on opposite sides of a cleavage site, wherein the domains are of different thermal stability after heating of the sample. The cleavage site may however be a known cleavage site. The cleavage site may be a caspase cleavage site. The cleavage site may be a known or unknown caspase cleavage site. The first and second signature peptides may be separated by a sequence of amino acids, for instance, at least 5, 10, 20, or 50 amino acids in the protein of interest. Preferably, the first signature peptide is an N-terminal peptide and the second signature peptide is a C-terminal peptide, or vice versa. N- and C-terminal here mean located closer to the N-or C-terminus, respectively, than the other signature peptide. It is not essential, though it is possible, that the N-and C-terminal peptides comprise the actual N- or C- terminal amino acid of the protein. The above descriptions of the first and second signature peptide apply mutatis mutandis to the first and second groups (or pools) of signature peptides discussed elsewhere herein. For instance, such groups are non-overlapping, are located on opposite sides of a cleavage site, may be separated by a sequence of amino acids in the protein of interest, and are preferably N- and C-terminally positioned, or vice versa. The signature peptides may be of unknown sequence. It is straightforward to determine the sequence of peptides detected by the present methods, and to identify them as component signature peptides of a particular protein in the proteome of a particular organism by reference to sequence databases. Proteins comprise a plurality of signature peptides, in fact many, signature peptides. Proteins of interest can be considered to comprise at least 4, 6, 8, 10, 12, 20, or more, signature peptides. As a minimum, the method of the invention comprises determining the level of a first signature peptide which is present in a first domain of the protein of interest, and the level of a second signature peptide which is present in a second domain of the protein of interest. The level of each signature peptide determined is the relative as compared to a control level of that signature peptide. However, the methods of the invention may comprise determining the levels of a plurality of signature peptides (e.g. at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 signature peptides) of the first domain, and a plurality of signature peptides (e.g. at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 signature peptides) of the second domain. The number of signature peptides assessed in the two domains need not be identical. Cleavage of the protein of interest is detected by a difference between the relative level of any one or more signature peptide(s) of the first domain and any one or more signature peptide(s) of the second domain. There is provided a method for detecting cleavage of a protein of interest in a non-purified test sample, wherein said protein of interest comprises a plurality of signature peptides present in a first domain and a plurality of signature peptides present in a second domain, wherein the method comprises the steps of: a) heating the non-purified test sample to provide a heated test sample; b) separating soluble from insoluble protein in the heated test sample of step a) to produce soluble and insoluble protein fractions of said heated test sample; and c) determining, in the soluble and / or the insoluble protein fraction of step b), the relative level of each signature peptide as compared to a control level, wherein cleavage is detected by a difference between said relative level of any one or more signature peptides of the first domain and the relative level of any one or more signature peptides of the second domain in the soluble protein fraction and / or by a difference between said relative level of any one or more signature peptides of the first domain and the relative level of any one or more signature peptides of the second domain in the insoluble protein fraction. Alternatively viewed, each protein of interest can be considered to comprise a first group (or “pool”) of signature peptides that are present in a first domain and a second group (or “pool”) of signature peptides that are present in a second domain, wherein cleavage of the protein of interest is detected by a difference between said relative level of any one or more signature peptides of the first group (or pool) and any one or more signature peptides of the second group (or pool) in the soluble protein fraction and / or by a difference between said relative level of any one or more signature peptides of the first group (or pool) and any one or more signature peptides of the second pool (or pool) in the insoluble protein fraction. The first and second "groups” or “pools” may each comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 signature peptides, and may comprise different numbers of signature peptides. Preferably, cleavage is determined by a difference between the levels of a plurality of signature peptides of the first domain (i.e. of the first group or pool) and the levels of a plurality of signature peptides of the second domain (i.e. of the second group or pool), e.g. between the levels of 2 or more, 3 or more, 4 or more or 5 or more signature peptides of each domain (i.e. of each group or pool). The combined (e.g. summed or averaged) relative levels of the plurality of signature peptides of the first domain (i.e. of the first group or pool) can be compared to the combined (e.g. summed or averaged) relative levels of the signature peptides of the second domain (i.e. of the second group or pool), wherein cleavage is detected by a difference between the combined (e.g. summed or averaged) level of the signature peptides of the first and second domains (i.e. the first and second groups or pools). Throughout this application, reference to a first and second signature peptide is intended to refer also to a plurality of signature peptides of such a first domain (or group or pool), and a plurality of signature peptides of such a second domain (or group or pool). As used herein, the term “test sample” is used to indicate a sample on which the steps of a method of the invention are performed. The steps of a method of the invention may also be performed on a “control sample”, however, this is not essential. The methods of the invention may comprise the comparison of data obtained using a test sample with control data (i.e. data obtained using a control sample) however, such control data may have been obtained previously. In other words, the data obtained with the control sample may be in the form of reference data, e.g. a reference database, and the methods of the invention need not comprise the performance of steps on a control sample. Data in this regard is preferably the level of one or more signature peptides, as described anywhere elsewhere herein. The methods of the invention are performed on non-purified test samples. Thus, the invention is concerned with analysis of impure samples. The methods of the invention are performed on a “non-purified” test sample, i.e. a crude or complex sample. Control samples as referred to herein are also non-purified, and the discussion herein in relation to test samples applies equally to control samples. As used herein, this means that the method is performed without the protein of interest having been purified. The sample is thus one that has not been subjected to a purification process that would result in the purification of the protein of interest. For instance, the sample has not been subjected to steps of extraction, precipitation and separation, e.g. by centrifugation or chromatography in order to purify the protein of interest. The term “non-purified sample” means that the protein of interest is present with other proteins and biomolecules in the sample. The term does not however exclude purification at the cellular level, i.e. separation or removal of a particular cell type or cell types from within the sample prior to the performance of the invention is possible. Prior to the heating step (step (a)), discussed below, non-purified samples may be prepared and / or processed. Such preliminary steps may comprise seeding cells in suitable media, treating cells with drugs or test molecules, incubating, washing, detaching, pelleting cells, re-suspending, etc. Such steps may also be comprised in the methods of the invention. None of these steps constitutes purifying the sample, because the protein of interest is still present in a non-purified form in or on the cells. Likewise, in the present methods, it is permitted to perform a step of cell resolution prior to any step of determining the level of a peptide. Such a cell resolution step may allow the analysis of the levels of peptide in particular cell types which have been isolated from a heterogenous sample (e.g. a tumour). In this respect, the invention provides a further step of cell sorting the samples, before or after the initial heating step. If the sample is a tumour sample, a step of tumour matrix degradation may also be necessary prior to any step of cell sorting. Methods for sorting cells are well known in the art e.g. FACS. Thus, the methods of the invention optionally comprise a step, prior to the peptide level determination step, and before or after the heating step, of cell sorting to isolate a cell type of interest from the sample. Cell selection, resolution, etc., e.g. cell sorting, results in a new non-purified sample for use in the steps of the method of the invention; the sample is still non-purified, as the term is used herein, because the protein of interest has not been purified, i.e. it is still present with other proteins and biomolecules in the sample. A non-purified sample may however comprise a heterogenous cell population. As mentioned above, the protein of interest is preferably used to detect proteins of interest that are within or on a cell. Thus, the non-purified sample preferably comprises intact cells. The non-purified sample preferably comprises a cell, e.g. comprises a plurality, i.e. population, of said cells (i.e. a “cell population”), that comprise the protein of interest. Preferred cells are as described above. Alternatively, however, the non-purified sample may not comprise intact cells. For instance, the non-purified sample may be a cell extract or lysate, e.g. a whole cell extract or whole cell lysate, or may be a urine or blood plasma sample. Cleavage of the protein of interest may be detected in such samples, and the protein of interest is not “within or on a cell” in such samples. The non-purified sample, may be a clinical or environmental sample. The non-purified sample may be obtained directly from a human or animal subject (e.g. patient). Preferably the sample is a patient sample. The non-purified sample may be a tissue sample (e.g. epithelium, muscle, nervous or connective tissue), blood sample, serum sample, plasma sample, lymph sample, cerebrospinal fluid sample, mucus sample, urine sample, or faeces sample, etc. The sample may be a liquid or solid tumour sample. The sample may also be obtained from a plant or part thereof, e.g. may be a plant extract. The non-purified sample may also be a cell colony or cell culture. A cell colony is a circumscribed group of cells, normally derived from a single cell or small cluster of cells growing on a solid or semi-solid medium (e.g. culture media with the addition of 0.1% or greater agar). The non-purified sample may be a liquid culture of cells. A liquid culture of cells may comprise cells which have all originated from a single cell i.e. the cells within the liquid culture may be clonal, or the liquid culture may comprise a suspension of different cells. The cells of the colonies or in liquid culture may be prokaryotic i.e. bacteria or eukaryotic cells e.g. yeast, unicellular eukaryotes such as Leishmainia, insect cells or mammalian cells or cell lines. Cells in liquid culture or grown as colonies may be formed as E.coli, Bacillus subtilis, Streptococcus lactis. Streptococcus lividens. Lactococcus lactis. Staphylococcus aureas. Aspergillus niger, Picia pastoris, Saccharomyces cerevisiae or Schizosaccaromyces pombe. The methods of the invention comprise heating the non-purified test sample. This produces (yields) a heated non-purified test sample (referred to herein simply as a heated test sample). Applying a temperature to a sample will cause melting / unfolding and subsequently precipitation of proteins that are thermally unstable at that temperature. CETSA assays are predicated on thermally unstable proteins being precipitated by the heating step, whereas proteins that are thermally stable at the heating temperature applied remain properly folded and so soluble. Hence, the solubility of a protein after the heating step is linked to its thermal stability. Thus, the detection of a soluble protein (e.g. in a soluble protein fraction) after heat treatment indicates that it is properly folded and thermally stabile at the heating temperature. The present inventors have surprisingly determined that CETSA can also be used to provide information on the thermal stability of individual protein domains after cleavage, not only full length proteins, in the sample, and that the sensitivity of the method is surprisingly high enough for differences in domain thermal stabilities to be detected, thereby allowing determination of the cleavage status of proteins, even in non-purified samples, which are complex and have a high level of background “noise”. A sample that has been heated according to the present methods will thus comprise a mixture of proteins and protein domains that are soluble (thermally stable at the temperature of heating) or insoluble (denatured by the temperature of heating). Thus, a "soluble” or “stable” protein or domain can be defined in reference to possession of a native or native-like conformation, or as one which remains soluble (e.g. remains in the supernatant after centrifugation of a heated non-purified sample). Likewise, an "insoluble" or “destabilized” protein or domain can be defined in reference to a loss of a native or native-like conformation, or as one which is not soluble (e.g. is not in the supernatant, but rather is in the insoluble protein fraction after centrifugation of a heated non-purified sample). It is possible that a protein or domain may unfold and precipitate at one specific temperature. It is also possible that a protein or domain may precipitate over a small temperature range. The “initial melting temperature” is the first temperature in the range at which the protein or domain precipitates, i.e. the first temperature at which the protein or domain is detected (or is detected at an increased level vs. control) in the insoluble fraction, and the temperature at which the first loss of soluble protein / domain is detected (e.g. is detected in the soluble fraction). Thus, the initial melting temperature is the lowest temperature at which a protein or domain begins to precipitate e.g. at least 5%, or at least 10%, of the protein or domain is precipitated. Conversely, the final melting temperature is the first temperature at which essentially no soluble protein or domain is detected (e.g. is detected in the soluble fraction.), or is the first temperature at which a decreased level vs. control is not detected, e.g. less than 5% of protein or domain is in soluble form. Therefore, when a protein or domain precipitates over a temperature range, the protein or domain may begin to precipitate or unfold at a particular temperature at which point the amount of soluble protein / domain present will begin to decrease and the amount of insoluble protein / domain present will increase (since thermal stability is linked to solubility). Therefore, some soluble protein / domain may still be detectable at the initial melting temperature until a slightly higher temperature is applied, at which point little or no soluble protein / domain is detectable. The temperature which can be applied in the present invention may be any temperature sufficient to cause the precipitation of the protein of interest or a domain thereof. The heating step may comprise heating the sample to a single temperature. The single temperature should be at or above the initial melting temperature of the protein of interest or a domain thereof. The initial melting temperature of the protein of interest or domain may be known. Even if the initial melting temperature of the protein of interest or domain is not known, the method may still comprise heating the sample to a single temperature (or at least one temperature) at or above the initial melting temperature of the protein of interest or domain thereof. A suitable temperature can be determined via routine optimisation. If after heating at a particular temperature no protein / domain of interest is identified in the insoluble protein fraction, or no loss of the protein / domain of interest is identified in the soluble fraction, then the initial melting temperature of the protein of interest or domain thereof has not been reached. Mammalian proteins and domains may begin to denature at 37°C, and a large majority will be expected to have begun to denature by 60°C. Therefore, the sample is preferably heated to a temperature above 37°C, preferably above 40°C, e.g. above 50°C. A higher or lower temperature may however be used if the protein of interest is not a mammalian protein, e.g. a higher temperature may need to be used if the protein is from a thermophilic bacterium. The skilled person will readily be able to determine suitable heating temperatures for their desired purposes. The methods of the invention may comprise repeated performance of the steps of the method using one or more different temperatures in said step of heating, wherein cleavage of the protein of interest is detected by a difference between said relative levels of said signature peptides after heating to at least one temperature. Preferably it is desirable to expose the test sample to a series of temperatures over a temperature range. A series of temperatures refers to at least two different temperature points, preferably at least three, four, five or six temperature points. A temperature range is the temperature range over which the protein of interest, or a domain thereof, unfolds and precipitates. The temperature range should include at least one temperature at or above the initial melting temperature of the protein of interest or domain thereof, which is as discussed above. The temperature points in the series of temperatures may differ from the adjacent temperature points by about 2, 3, 4, 5, 6, 7, 8, 9 or 10 °C, and the difference between temperature points need not be constant. Preferably, the heating step comprises heating the non-purified sample to a series of temperatures in the temperature range of 30 to 80°C, more preferably 35 to 70°C, more preferably 37°C to 60°C, 37°C to 59°C, or 37°C to 58°C, e.g. 40°C to 55°C. Measurements may be taken at any interval, but typically at one degree increase, two degree increase or three degrees increase in temperature. The sample may be heated to any temperature, e.g. at any one of more of 37, 38 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 and 59°C, as long as one of the temperatures is equal to or higher than the initial melting temperature for the protein of interest of domain thereof. Temperatures below 37°C and above 59°C may also be used when determined suitable by the user of the method; as mentioned above, this is within the general competencies of the person of ordinary skill in the art. Preferred temperature series comprise heating to temperatures of i) 37°C, 47°C, 50°C, 52°C, 54°C and 57°C; ii) 37°C, 47°C, 50°C, 53°C, 55°C and 59°C; or iii) 37, 42, 46, 52 and 58°C. Where the sample is heated over a temperature range, this can be carried out in a PCR machine where an initial temperature can be set and then increased by the desired amount after a particular amount of time e.g. 1, 2, 3, 4 or 5 minutes. However, incubators, waterbaths, etc. may also be used. When aliquots of the sample are taken before the aliquots are heated, they can be placed in different positions in a PCR heating block, with the different desired heating temperatures used in the different positions. The heating step comprises holding the sample (or aliquot thereof) at the desired temperature for a desired period, which may preferably be 20 seconds to 5 minutes, preferably 1, 2, or 3 minutes. The precise holding time depends on the volume of the sample (or aliquot) being heated, and it is within the competencies of the person of ordinary skill in the art to determine an appropriate holding time for their purposes. A preferred holding time is about 3 minutes. When the heating step comprises heating the sample (e.g. the test sample) to a series of temperatures in a temperature range, the result is to obtain (produce) a plurality of heated sample aliquots. Thus, preferably the heating step comprises obtaining a plurality of heated sample aliquots by i) heating the non-purified test sample to a series of temperatures in a temperature range and obtaining an aliquot of the sample after heating at each temperature, or ii) obtaining a plurality of non-purified test sample aliquots and heating each sample aliquot to a different temperature within a series of temperatures in a temperatures range to obtain a plurality of heated test sample aliquots. In this way, the plurality of heated sample aliquots are the product of the heating step. The number of individual samples used in the heating step may depend on the number of different temperatures which it is desired to expose the sample to. In this instance, each sample aliquot is heated, or has been heated, to only one particular temperature. Each heated sample aliquot may then be processed separately as set out in the subsequent steps of the method. This permits determination of the level of signature peptides in the soluble and / or insoluble protein fraction at each temperature point of the series of temperatures used. Cleavage of the protein of interest is then detected by a difference between said relative levels of signature peptides as described above, in the soluble and / or insoluble protein fraction of one or more of the heated sample aliquots i.e. at one or more temperatures (i.e. temperature points) in the series of temperatures used. Thus, preferably, the methods of the invention comprise a) heating the non-purified test sample, wherein said heating step comprises i) heating the non-purified test sample to a series of temperatures in a temperature range and obtaining an aliquot of the sample after heating at each temperature, or ii) obtaining a plurality of non-purified test sample aliquots and heating each sample aliquot to a different temperature within a series of temperatures in a temperature range, to obtain a plurality of heated test sample aliquots; b) separating soluble from insoluble protein in each of the heated test sample aliquots to produce soluble and insoluble protein fractions thereof; and c) determining, in the soluble and / or the insoluble protein fraction of each heated test sample aliquot, i) a first relative level, which is the level of the first signature peptide as compared to a control level, and ii) a second relative level, which is the level of the second signature peptide as compared to a control level, wherein cleavage is detected by a difference between said first and second relative levels in the soluble protein fraction of at least one heated test sample aliquot and / or by a difference between said first and second relative levels in the insoluble protein fraction of at least one heated test sample aliquot. Preferably, the protein of interest comprises a plurality of signature peptides in said first domain, and a plurality of signature peptides in said second domain, and said method comprises a) heating the non-purified test sample, wherein said heating step comprises i) heating the non-purified test sample to a series of temperatures in a temperature range and obtaining an aliquot of the sample after heating at each temperature, or ii) obtaining a plurality of non-purified test sample aliquots and heating each sample aliquot to a different temperature within a series of temperatures in a temperature range, to obtain a plurality of heated test sample aliquots; b) separating soluble from insoluble protein in each of the heated test sample aliquots to produce soluble and insoluble protein fractions thereof; and c) determining the relative level of each signature peptide in the soluble protein fraction of each heated test sample aliquot, as compared to a control level of said signature peptide, and / or determining the relative level of each signature peptide in the insoluble protein fraction of each heated test sample aliquot, as compared to a control level of said signature peptide; wherein cleavage is detected by a difference between the relative level of any one or more signature peptides of said first domain and the relative level of any one or more signature peptides of said second domain, in a protein fraction of at least one heated sample aliquot. An “aliquot” of the sample may be of any volume, e.g. 1 or 2 pl. If the sample in question is a cell colony, rather than a liquid sample, then a portion of the colony may be lifted off after each incubation e.g. by placing filter paper on the top of the colony. Thus the invention may comprise heating the sample to a series a range of temperatures and processing and analysing an aliquot taken after incubation at each temperature in order to produce a precipitation curve for each signature peptide. Optionally, the plurality of heated sample aliquots obtained may be pooled to form a pool of heated sample aliquots, and said pool of heated sample aliquots is thereby the product of the heating step that is processed as set out in the subsequent steps of the method. Throughout this application, any reference to a step being performed on, with or to a heated sample is also to be considered as a reference to said step being performed on, with or to an aliquot thereof, i.e. on, with or to a heated sample aliquot or a pool thereof. The methods of the invention may comprise a step of lysis before or after the heating step. However, if present then the lysis step is preferably performed after the heating step has been performed. Thus, preferably the sample heated in the heating step contains intact cells. A lysis step is a cell lysis step, i.e. a step of lysing cells in the sample. Such steps are well-known in the art. If a lysis step is performed prior to the heating step, then it is important that the lysis step is non-denaturing, allowing proteins and domains in the sample to retain a native i.e. correctly folded or native-like conformation. This is referred to herein as native lysis. This can be carried out chemically or otherwise using reagents which are well known in the art e.g. mild buffers such as PBS or Tris at physiological pH. The degree of lysis must be sufficient to allow proteins of the cell to pass freely out of the cell. Typically, when dealing with membrane bound proteins or domains, lysis is performed in the presence of detergents or amphiphiles, for example Triton X-100 or dodecylmaltoside, to release the protein or domain from the membrane. The lysis step can alternatively be carried out by freeze thawing the cells or colonies, e.g. in liquid nitrogen. More preferably, lysis is carried out using both native lysis buffer and freeze thawing the cells. The lysis buffer may contain lysozyme, for example at 50-750 pg / ml, or at 100-200 pg / ml. DNAse can also be found in native lysis buffer preferably at 250-750 pg / ml. Native lysis buffer may contain for example 20mM Tris, pH 8, lOOmM NaCl, lysozyme (200pg / ml) and DNAse I (750 pg / ml). Preferably, lysis is performed in the presence of one or more protease inhibitors, which are well-known and widely available. Typically, the cells will be exposed to the lysis buffer for 15-60 minutes, preferably around 30 minutes. The step of freeze thawing is preferably repeated, i.e. two or more cycles, preferably 3 or more cycles of freeze thawing are performed. Preferably, lysis is achieved by a 30 minute incubation at room temperature with lysis buffer and three xlO minutes freeze thawing. A step of lysis may be performed after the heating step. The step is preferably performed before separation of the heated sample (or aliquot) into soluble and insoluble fractions. Again important that the lysis step is non-denaturing (i.e. native lysis), allowing proteins and domains in the sample to retain a native i.e. correctly folded or native-like conformation. . If present, a lysis step preferably occurs before the step of determining the soluble / insoluble level of the protein / peptide of interest. If present, a lysis step preferably occurs prior to any step of separating soluble from insoluble protein in the heated test sample to yield soluble and insoluble protein fractions. If the protein of interest or domain thereof is within a cell, and if a lysis step is not performed prior to the heating step, then a lysis step is preferable after the heating step. However, lysis is optional; the detection of soluble / insoluble proteins and domains may be performed within intact cells, e.g. when a cell is fixed and peptide / protein-specific imaging probes recognising only the soluble protein or domain, or alternatively only precipitated protein or domain, are applied thereto. If fixing cells, it is important that the fixing method used preserves proteins in their native stage. One suitable fixation method comprises the application of formaldehyde (e.g. 4% formaldehyde). If the protein of interest or domain is inside the cell, then the cell needs to be permeabilized for the probe to access the protein / domain. Permeabilization can be achieved using detergents e.g. NP-40 or triton-x 100. The proteinspecific or domain-specific probe may be an antibody conjugated to a fluorophore as described elsewhere herein. Quantification of the level of the probed protein or domain can be performed, e.g. by imaging in a fluorescence microscope or by flow cytometry / FACS. When the present method is carried out on cell colonies, lysis may be carried out directly on those colonies i.e. there is no need to pick the colonies and grow them in liquid culture (although this can be done). In this instance, it is preferred that the separation step is one of filtration. Further, where the method is performed on cell colonies, preferably, the filter paper is overlayed on the colonies to lift the colonies from the semi-solid or solid growth media. Alternatively, filters could be placed on the growth media and cells seeded directly onto the filter, the filter could then simply be lifted off with the colonies already on it. Preferably, the lifting of the colonies in this way can be carried out prior to the lysis step. As indicated above, the lysis can be carried out directly on the colonies on a filter. The filter with colonies attached can be treated with lysis buffer or overlaid on other membranes / filters treated with lysis buffer Typically, the percentage of cells lysed within a sample (e.g. a cell colony or cell culture) during the lysis step is 5-100%. Thus, it is not necessary when performing a step of lysis for all cells within a sample to be lysed. The methods of the invention preferably comprise a step of separating soluble from insoluble protein in the product of the heating step (i.e. in the heated sample or aliquots thereof), to produce soluble and insoluble protein fractions of said sample, and then determining the relative levels of the signature peptides, as described elsewhere herein,in either or both of the soluble and insoluble protein fractions. This separation step achieves the physical separation of the proteins and domains that remain stable (i.e. are in the soluble fraction) from those that are destabilized (i.e. are in the insoluble fraction) after the heating step. Stable as used herein means “thermally stable”, i.e. not denatured or “destabilized” due to the heating of the non-purified sample in the earlier step. The separation step b) is a simple step of separating soluble from insoluble proteins and domains to produce (i.e. obtain) soluble and insoluble proteins fractions after heating. Soluble proteins and domains are associated with being thermally stable, i.e. correctly folded, at the temperature applied to the sample, whereas insoluble proteins and domains are not thermally stable, and are precipitated by the temperature applied to the sample. The separation step is therefore to discriminate between soluble and insoluble proteins and domains. The separation step can involve any separation method which is capable of separating soluble from insoluble protein (i.e. proteins and domains). For example, a step of centrifugation, chromatography or filtration may be used. Preferably the separation step is centrifugation or filtration. Any suitable centrifugation step for separating soluble and insoluble protein in a heated sample may be used, and such steps are within the general competencies of the person of ordinary skill in the art. Centrifugation can typically be carried out between 100g and 50000g. Centrifugation is preferably carried out above 2000g, e.g. between 2000g and 50000g, or between 2000g and 20000g. The duration of centrifugation can be from 1 minute (typically at least 10 minutes) to at least 1 hour, where the duration required generally decreases as the centrifugal force increases. Particularly suitable conditions for providing only soluble proteins and domains in the resultant supernatant include 30 minutes at 3000g or 20 minutes at 20000g. A filter can be used to separate soluble from insoluble proteins and domains where soluble proteins and domains will pass through a filter. Standard filter membranes can be used for filtering heated samples where the filters will typically have a pore size from 0.015 pm to 12 pm, preferably from 0.35 to 1.2 pm, more preferably from 0.45 pm to 0.8 pm. Preferably the filters have pore sizes below 4.0 pm, typically below 2.0 pm, more preferably below 1.0 pm. Suitable filters are Super and GH polypro (from Pall) and Nucleopore (From Whatman). It will be appreciated that proteins of interest and domains thereof from eukaryotic and prokaryotic samples and from different cell types may require the use of filters with different pore sizes. Selection of a suitable filter is well within the competency of someone skilled in this field. For example, it is possible to select an appropriate pore size, by using a set of test proteins for the desired cell type or sample and investigating their behaviour with filters of varying pore sizes. Proteins and domains can simply be allowed to pass through the filter, possibly as a result of capillary action. Alternatively, force may be applied vertically on the filter paper, wherein such forces can include the application of pressure or vacuum. According to the methods of the invention it is possible to analyse either (or both) the insoluble or soluble protein fractions in the subsequent method steps. Thus the relative levels of the signature peptides may be determined in either or both of the soluble and insoluble protein fractions. In all aspects and embodiments, the methods of the invention preferably comprise determining and comparing the relative levels of signature peptides (or groups or pools thereof) in the soluble protein fraction of the heated test sample / heated test sample aliquots. In other words, preferably the soluble protein fraction of step b) is analysed. Hence, if step b) comprises a centrifugation separation step, then the supernatant can be analysed to determine the relative levels of the signature peptides. If step b) comprises a filtration separation step, then the filtrate can be analysed to determine the relative levels of the signature peptides. If the insoluble fraction is analysed, then it is preferably solubilised prior to analysis. Steps for solubilizing an insoluble protein fraction are well-known in the field, and any suitable solubilization step may be performed. For example, the insoluble proteins and domains in the insoluble protein fraction may be solubilized with detergents or combinations of detergents, including sodium dodecyl sulfate, thiol reducing agents (such as 2-mercaptoethanol and dithiothreitol) and chaotropic agents such as urea, etc. The protein fraction may be subsequently dissolved in loading buffer prior to application to separation gels. It is important to note that if an insoluble fraction is solubilized for analysis, the level of signature peptides in the insoluble form of the protein or domain in the heated test sample is still being determined due to the previous separation of the insoluble and soluble protein fractions. The methods of the invention comprise determining at least the relative level of a first peptide as compared to a control level, and the relative level of a second signature peptide as compared to a control level. As explained above, these levels may be determined in the soluble and / or insoluble protein fraction of said heated test sample (or heated test sample aliquot). By “determining the level” of a peptide means determining the abundance of the peptide in the sample, aliquot or fraction concerned. Determining the level of a peptide may comprise quantification of the peptide, i.e. quantitative determination of the amount or concentration of the peptide. The determination of the level of a peptide is the relative level of the peptide as compared to a control level, which is the level of the same peptide in a control sample. As explained below, mass spectrometry is a preferred method for determining the level of signature peptides. It is within the general competencies of the person of ordinary skill in the art to interpret peaks in mass spectrometry data to determine the level of peptides of interest therein. Many well-known and widely available computational tools exist for this purpose, and any suitable tool may be used. The relative level of a signature peptide is the difference between the determined level of the signature peptide in protein fraction of a heated test sample (or aliquot thereof) and the level of that signature peptide in an equivalent protein fraction of a heated control sample (or aliquot thereof). Determining the level of the signature peptides may be performed by any suitable technique. Preferably, the technique does not involve the labelling or tagging of the protein of interest prior to the performance of the heating step (e.g. step a) However, tagging or labelling of the protein of interest in the cell before the heating step may be performed, and may be desirable in some instances, e.g. to assist with quantification of the protein after heating (and optionally lysis) e.g. with an affinity reagent. Tagging and labelling of the protein after heating step (step a)) has been performed is also possible. The levels of the signature peptides may be determined by mass spectrometry or affinity binding. Affinity binding is discussed elsewhere herein in relation to determination of levels of domains, and the discussion applies mutatis mutandis to the detection of the signature peptides. Preferably, the levels of the signature peptides are determined by mass spectrometry. Mass spectrometry is particularly preferred when the levels of many peptides are to be determined, e.g. in methods in which levels of a plurality of signature peptides, e.g. groups (or pools) of signature peptides, are to be determined, or when levels of each signature peptide in the sample / aliquot / fraction are to be determined. Mass spectrometry analysis typically requires a separation step as described above to be performed and the subsequent analysis of the soluble fraction, or of the insoluble fraction, as described above. Mass spectrometry analysis of an insoluble protein fraction requires a step of solubilization of the fraction prior to analysis, as described above. Preferably, step c) of the method preferably comprises subjecting either or both the soluble and insoluble protein fractions of step b) to mass spectrometry analysis to yield mass spectrometry data, and analysing the mass spectrometry data to determine the levels of the first signature peptides. Methods of mass spectrometry, and analysis of mass spectrometry data to determine levels of protein / peptide sequences therein, are well known in the field, and any suitable method may be used. Preferred methods are described in the Examples. Semi-quantitative or preferably quantitative mass spectrometry (MS) may be used. A preferred method is liquid chromatography MS (LC-MS). Mass spectrometry methods can be used to measure a large fraction of the proteome, but can also be targeted to detect only a smaller number of signature peptides, if desired. Steps of sample preparation for mass spectrometry analysis are well-known in the field and any such steps may be used. It is within the competencies of the person of ordinary skill in the art to prepare samples for mass spectrometry analysis. Preferred methods are described in the Examples. The methods thus may comprise a step of preparing the protein fraction(s) of step b) for mass spectrometry analysis. When performing mass spectrometry on protein-containing samples it is typical to digest proteins in the sample (or protein fraction thereof) into smaller fragments (i.e. peptides). Thus, preferably the methods of the invention comprise a step, after step b), of digesting protein (e.g. said domains) in said soluble and / or insoluble protein fraction to produce at least a first signature peptide and at least a second signature peptide as defined herein. Preferably a plurality of signature peptides, as defined here, is produced. Preferably said step of digesting proteins is a step of proteolysis more preferably trypsinisation. The step generates signature peptide fragments of said domains. Thus, digestion in the protein fraction is preferably via proteolysis, preferably via trypsinisation, i.e. trypsin digestion). For instance, trypsinization may be performed at 37°C, e.g. overnight. Preferred methods are as described in the Examples. The proteins in the protein fraction may be denatured prior to digestion. Thus, the soluble and insoluble protein fractions obtained in step (b) comprise intact proteins and protein domains, which are either correctly folded and soluble, or denatured and insoluble, respectively. Particularly prior to mass spectrometry analysis, the proteins and domains in the fractions are digested (e.g. trypsinized) to produce peptides (i.e. peptide fragments of said proteins and domains). Many of these peptides will be signature peptides of the protein of interest, present in the first or second domain of the protein. The levels of these signature peptides are then determined via mass spectrometry (or by affinity binding if desired). The levels of the signature peptides in the soluble and / or insoluble fraction provide information on the levels of the domains from which they are derived in those fractions, and so on the thermal stability of those domains in the heated sample (or aliquot). After digestion, the resulting peptides may be labelled to facilitate detection by mass spectrometry. Again, MS-labelling strategies are well known in the art, and any suitable method may be used. Preferred methods of labelling include ITRAQ, or Isobaric Tandem Mass Tags (TMT), e.g. TNT-lOplex or (TMTpro)-16plex or 18-plex. The TMT-lOplex or higher-plex strategy is preferred as it permits multiple replicates of both control and test samples to be measured and normalized for each sample in the same experiment. Prior to MS measurement, samples (e.g. comprising labelled trypsinisation product peptides) may be pre-fractionated, e.g. via liquid chromatography. Suitable techniques are well known in the field. The MS measurement would be performed using a mass spectrometry instrument, such as a Orbitrap Q Executive or Fusion (Thermo-Fisher Scientific). Full scan MS spectra may be acquired, e.g. in the range of 375-1500 m / z. Mass spectrometry analysis results in mass spectrometry data, which can be analysed by well-known and commonly used data processing programs to indicate the levels of each of the peptide fragments (including each of the signature peptides) present in the analysed sample. The identity (i.e. amino acid sequence) of the signature peptides need not be known, as mass spectrometry methods typically permit detection and determination of the levels of high numbers of peptides / proteins in a single assay / analysis. For instance, in a fourier transform ion cyclotron resonance experiment using an orbitrap instrument, typically 1000-10 000 peptides / proteins can be detected simultaneously in a complex sample (e.g. a cell lysate). The level of a signature peptide determined as part of the methods of the invention is a relative level, i.e. the difference in the level of the signature peptide in a protein fraction of a test sample as compared to in a corresponding protein fraction of a control sample. If, at a given temperature point, the amount of signature peptide detected in the test sample fraction is different (i.e. has “shifted”) as compared to the amount in the corresponding control sample fraction, then the conditions of the test sample have caused a “stability shift” for the protein domain from which the signature peptide is derived. The relative level of a signature peptide (i.e. its “shift”) in a protein fraction can also be expressed in the form of a ratio or “fold change”. These provide numerical values for the difference between the level of the signature peptide in the protein fraction of the heated test sample (or aliquot)) as compared to the level of the same signature peptide in corresponding fraction of a heated control sample (or aliquot). Thus, a ratio or “fold change”, indicates the extent of the difference between the test and control levels for a given signature peptide. The ratio may be expressed as the ratio of the test level of the signature peptide to the control level of the signature peptide, or vice versa. The fold change (or ratio) may be expressed as the test level of the signature peptide divided by the control level of the signature peptide, or vice versa. The fold change may be expressed on a log scale (“log fold change” or “log FC”, expressed using any desired base unit. Preferably, the fold change is expressed as a "log2 fold change", which is calculated according to the formula log2(FC) = log2 Q) = log2(a) — log2(b), wherein a and b are the test and control levels of the signature peptide concerned, respectively As explained above, the test and control levels may be determined computationally, e.g. from mass spectrometry data. Software for determining and displaying the ratio and / or fold changes discussed above is also widely available. Measurement of individual protein abundance may be computed from raw mass spectrometry data using proteomics software, which is well-known and widely available, such as Proteome Discoverer or MaxQuant. The data obtained may be normalized, e.g. by log2 transformation. The log2 fold changes can then be obtained by computing the difference between test and control samples. The present inventors have determined that if there has not been cleavage of the protein of interest, then the relative levels of two or more signature peptides from two different domains of the protein (each as compared to a control level) in either or both of the soluble and insoluble fractions after heating will be essentially the same. This is because the two domains have not been physically separated, and so their thermal stabilities are linked; they are both still present as part of the protein of interest, and so they will appear in the soluble / insoluble fractions after heating to the same extent as each other. Thus, those domains (and signature peptides from those domains) are present at essentially the same level as each other in the soluble fraction, and in the insoluble fraction, after the heating step. However, the present inventors determined that if the protein of interest is cleaved in the test sample, then the first and second domains may have different thermal stabilities, i.e. can fold (i.e. be soluble) or misfold (i.e. be insoluble) at different temperatures, and so signature peptides of the first domain would be detectable at different relative levels (vs. control) to the relative levels of signature peptides from the second domain, in the insoluble and / or soluble protein fraction in a CETSA assay. The present inventors determined, surprisingly, that CETSA-based assays are sufficiently sensitive to detect such differences in thermal stabilities of protein domains, thereby permitting detection of protein cleavage. Cleavage of the protein of interest is determined by a difference between the relative levels of two signature peptides from different domains of the protein (e.g. said first and second relative levels) because such a difference indicates that the two signature peptides are present in domains having different thermal stabilities in the test sample. In other words, if, after heating, the relative levels of signature peptides from the two different domains of a protein of interest (each peptide level being that as compared to a control level) are different in either or both of the soluble and insoluble fractions, then the protein of interest is determined as being cleaved in the test sample. This is because protein cleavage has resulted in the two domains being physically separated, and so independent in terms of their thermal stability, which is not the case in an non-cleaved protein. Thus if cleavage has occurred, then after heating to a given temperature, one domain may still be correctly folded and soluble to a greater extent than the other. Thus, the two domains (and signature peptides from those domains) may be detected at different levels either in the soluble fraction, and / or in the insoluble fraction, after the heating step. Without wishing to be bound by theory, the present inventors considered that after cleavage, different protein domains may display different thermal stabilities due to, for instance, having different interactions and associations, for instance experiencing differing steric constraints, differing associations with protein binding partners, etc.. The present inventors realised that this might lead to different levels of signature peptides from different domains of the protein being detectable in the soluble and / or insoluble protein fraction of a heated sample if the protein was cleaved, but not in the absence of cleavage. However, it had not been demonstrated, and it was not expected that a CETSA assay would have the sensitivity necessary to detect these different levels of protein cleavage products in a nonpurified sample. CETSA had only been previously been demonstrated at the intact protein level. As shown in the Examples, the present inventors have surprisingly demonstrated that the CETSA-based methods of the invention do have the sensitivity to determine differences in peptide levels from protein cleavage products with differing thermal stabilities, in complex samples, and so can detect the cleavage status of proteins in the sample. Accordingly, cleavage of the protein of interest in the non-purified test sample is detected by a difference between at least two relative levels, e.g. between the first and second relative levels described above, i.e. a first relative level, which is the level of the first signature peptide as compared to a control level, and a second relative level, which is the level of a second signature peptide as compared to a control level, said first and second signature peptides being present in a first and second domain of the protein of interest in the non-purified test sample, respectively. If the relative levels of a plurality of signature peptides of each of the first and second domains are determined, then cleavage is detected by a difference between the relative level of one or more signature peptide(s) of the first domain and the relative level of one or more signature peptide(s) of the second domain. The combined (e.g. summed or averaged) relative levels of a plurality of signature peptides of the first domain may be compared with the combined (e.g. summed or averaged) relative levels of a plurality of signature peptides of the second domain, wherein cleavage is detected by a difference between the two combined (e.g. summed or averaged) relative levels. In methods in which a plurality of heated sample aliquots is obtained, each obtained after heating to a different temperature in a temperature series, the relative level of the first and second signature peptides, or the relative level of each of a plurality of signature peptides of the first and second domains, may be determined in the soluble and / or insoluble protein fraction of each heated sample aliquot (i.e. after heating to each temperature). Cleavage is then detected by a difference between the above-mentioned relative levels in either or both of the soluble and insoluble protein fractions of at least one heated test sample aliquot. Again, the difference may be the difference between the combined (e.g. summed or averaged) relative levels of the signature peptides of the first domain and the combined e.g. (e.g. summed or averaged) relative levels of the signature peptides of the second domain, in in either or both of the soluble and insoluble protein fractions of at least one heated test sample aliquot. For any given signature peptide, the relative level of the signature peptide determined in the soluble or insoluble protein fraction of each heated sample aliquot, i.e. at each temperature, may be combined (e.g. summed or averaged) to provide a temperature-combined relative level, e.g. a temperature-summed relative level or a temperature-averaged relative level. Cleavage may then be detected by a difference between the temperature-combined relative level of the first signature peptide and the temperature-combined relative level of the second signature peptide, or by a difference between the temperature-combined relative level(s) of one or more signature peptides of the first domain and the temperature-combined relative level(s) of one or more signature peptides of the second domain. Thus, step c) may further comprise calculating a temperature-summed relative level of the first signature peptide, which is the sum of the first relative levels determined in the soluble protein fractions of each heated test sample aliquot, and calculating a temperature-summed relative level of the second signature peptide, which is the sum of the second relative levels determined in the soluble protein fractions of each heated test sample aliquot in each protein fraction, and / or calculating a temperature-summed relative level of the first signature peptide, which is the sum of the first relative levels determined in the insoluble protein fractions of each heated test sample aliquot, and calculating a temperature-summed relative level of the second signature peptide, which is the sum of the second relative levels determined in the insoluble protein fractions of each heated test sample aliquot in each protein fraction, wherein cleavage is detected by a difference between the temperature-summed relative levels of the first and second signature peptides in either or both of the soluble and insoluble protein fractions. In methods in which relative levels of a plurality of signature peptides of the first and second domains are determined, step c) may further comprise calculating a temperature-summed relative level for each signature peptide, which is the sum of the relative levels of the signature peptide determined in the soluble protein fraction of each heated test sample aliquot, wherein cleavage is detected by a difference between the calculated temperature-summed relative level of one or more signature peptides of said first domain and the calculated temperature summed relative level of one or more signature peptides of said second domain; and / or ii) calculating a temperature-summed relative level for each signature peptide, which is the sum of the relative levels of the signature peptide determined in the insoluble protein fraction of each heated test sample aliquot, wherein cleavage is detected by a difference between the calculated temperature-summed relative level of one or more signature peptides of said first domain and the calculated temperature summed relative level of one or more signature peptides of said second domain. If desired, the temperature-combined relative level(s) of a plurality levels of peptides from the first domain can be combined (e.g. summed or averaged), and the temperature-combined relative level(s) of a plurality levels of peptides from the second domain can be combined. This provides a temperature-combined relative level for each of the two protein domains, rather than for individual signature peptides therein. Cleavage may then be detected by a difference between the first and second combined temperature-combined relative level(s). Each of these differences is indicative of cleavage. Each of these key differences may be between relative levels determined in either the soluble fraction of a heated test sample (or aliquot) and / or in the insoluble fraction of a heated test sample (or aliquot). Preferably the relative levels are indicated as a fold change. In all aspects and embodiments, the difference between the relative levels of the signature peptides that are indicative of cleavage is a measurable difference, preferably a significant difference, preferably a statistically significant difference. Appropriate methods of determining statistical significance are well known and documented in the art and any of these may be used. Such methods may be based on calculation of P values or alternatively standard deviation or related methods using replica measurements. In any statistical analysis referred to herein, preferably the statistically significant difference over the control level has a probability (P-value) value of <0.1, preferably <0.05. The present CETSA assays are based on thermal stability changes in a protein domain, wherein heating results in altered levels of the soluble and insoluble forms of the protein domain (e.g. in the soluble and insoluble protein fractions of the sample) as compared to in heated control samples. But the amount of the domain is the sample is not altered. The observed differences in the level of signature peptides of a domain as compared to control levels are not due to increased expression of the domain; this can be confirmed / controlled via standard quantitative proteomics, which is within the competencies of the person of ordinary skill in the art. The present inventors have determined that when a “shift” vs. control at one or more temperature points is observed for one or more signature peptides from a first domain is different from a “shift” vs. control at the same temperature points for one or more signature peptides from a second domain, then this is due to the protein of interest being cleaved in the test sample. If a difference between the level of a signature peptide in a protein fraction of the test sample as compared to in a control sample is observed, then typically it is observed at temperature points above the initial melting point of the protein of interest or domain thereof, but not necessarily at lower temperatures. As explained above, the determination of the level of a signature peptide comprises determining the relative level of the signature peptide as compared to a control level, which by definition is a control level of the same signature peptide. This is the case for all signature peptides assessed. A control level is the level of the signature peptide determined in a corresponding (or equivalent) protein fraction of a heated control sample (or aliquot). The control sample is a nonpurified control sample. The control level is determined in a heated protein fraction of a nonpurified control sample (or aliquot), using the same (corresponding) method steps as used to determine the level in the test sample. The person of ordinary skill in the art understands that comparison to a control level means comparison of the level of same signature peptide in the same (corresponding) protein fraction type (i.e. insoluble or soluble protein fraction) of a heated control sample (or aliquot). By way of example, the level of a signature peptide in the soluble protein fraction of the heated test sample (or aliquot) would be compared to the level of the same signature peptide in the soluble protein fraction of the heated control sample (or aliquot), and the level of signature peptide in the insoluble protein fraction of the heated test sample (or aliquot) would be compared to the level of the same signature peptide in the insoluble protein fraction of the heated control sample (or aliquot). Thus, when a soluble fraction of a heated test sample is analysed, the soluble fraction of the heated control sample is also analysed. Further, insoluble fractions are compared with each other from the heated test and control samples. Preferably the control, i.e. the control sample / aliquot / fraction, referred to for determining the relative level of a first signature peptide is the same control, i.e. the same control sample / aliquot / fraction, referred to for determining the relative level of further signature peptides. It is within the competencies of the person of ordinary skill in the art to identify a suitable control sample. A control sample is also a non-purified sample. The control sample is preferably of the same or similar type (i.e. corresponding) type as the test sample, unless differences in the sample type are to be analysed for their effects on cleavage of one or more proteins of interest. Thus, for example, if a tissue sample from a tumour is used as the test sample, preferably the same tissue sample type is used as the control sample, e.g. a tissue sample from a tumour. If the two samples are subject or patient samples, then they are preferably obtained from the same subject or patient. They may be obtained from the same subject or patient at different times. Furthermore, both the test and control samples will preferably have been exposed to the same or similar heating and separation steps e.g. heated at the same temperature(s) or a at similar temperature(s), e.g. within 0.5-2°C, and / or treated with the same separation, lysis and other steps, if performed. The test and control sample may comprise the same sample at different time points, or may be subject samples obtained at different time points. The test and control samples may comprise the same cell types, unless it is desired to analyse cleavage of one or more proteins of interest across differing cell types. Thus, the test and control samples are largely similar or identical, and have been similarly or identically treated. However, the test sample differs from the control sample in one respect, or has been treated differently in one respect, such that a comparison can be made between the test and control samples in order to analyse the effects of that difference on protein cleavage. The person of ordinary skill in the art understands that a test sample by definition comprises a feature (or component) that is absent from a control sample, or has been exposed to a condition or treatment step to which the control sample has not been exposed, or vice versa. Thus, preferably, the test sample has been exposed to a test condition, to which the control sample has not been exposed. Thus, the cleavage detected by the methods of the invention is preferably cleavage due to, or induced by, said test condition. Alternatively viewed, preferably the test sample has been exposed to a first (test) condition and the control sample has not been exposed to said first (test) condition. By way of non-limiting example, the test sample and the control sample may have been differentially treated; exposed to different conditions; obtained from different subjects, different cell cultures or different tissue cultures; or obtained from the same subject, cell culture or tissue culture at different time points. The test sample and the control sample may comprise cells of a different genotype or phenotype. Optionally: i) the test sample has been exposed to a test molecule (e.g. a drug, a physiological metabolite or a cytokine) and the control sample has not been exposed to said test molecule or vice versa', ii) the test sample has been obtained from a drug-treated subject and the control sample has been obtained from a subject that has not been drug-treated or vice versa', iii) the test sample and the control sample have been treated with different drugs or are obtained from subjects that have been treated with different drugs; iv) the control sample and the test sample have been obtained from the same subject at different time points; v) the control sample and the test sample have been obtained from the same subject before and after drug treatment, respectively, or vice versa. vi) the test sample is a patient sample and the control sample is a healthy control sample or vice versa, vii) the test sample and control sample are different biological samples or comprise different cell lines; viii) the test sample is a diseased sample and the control sample is a healthy control sample or vice versa, ix) the test sample and control sample are obtained at two different time points from the same cell culture or tissue culture; or x) the test sample comprises cells comprising a genetic modification and the control sample comprise cells lacking said genetic modification, or vice versa. Thus, the test condition may be treatment with a test molecule, and is preferably drug treatment. The "test molecule" as used herein may be a protein, polypeptide, peptide, RNA, or DNA molecule. The test molecule may be a drug or pharmaceutical product, a cell metabolite or a hormone e.g. in serum. The test molecule may be naturally occurring or may be synthetically or recombinantly produced. The test molecule may alternatively be a test composition, e.g. comprising a mixture of molecules as defined herein. Plant extracts are an example of a preferred test composition. The test condition may also be that further (different) cells are added to the test sample, i.e. the cells in the test sample are exposed to cells of a further (different) type, such as immune cells, in order to detect protein cleavage induced by said further cells. Preferably, the added further cells are washed off before any step of lysis step is performed. Alternatively, the added further cells may have been grown in labelled amino acids, which results in the proteins and peptides of such further cells having different molecular weights from those in the cells of the test sample. This allows signature peptides from the different cell types to be distinguished by mass-spectrometry. Preferably, the method of the invention comprises a step of exposing the test sample to said test condition, said step being performed prior to the heating step (e.g. step a)). Preferably, the test condition is a step of adding (i.e. administering) a test molecule, as defined anywhere herein, to the non-purified test sample, i.e. a step of exposing the cells (cell population) within the non-purified test sample to the test molecule. Preferably this is an in vitro step. Thus, preferably, the non-purified test sample is a sample which has been exposed to, i.e. to which has been added (i.e. administered), a test molecule as defined anywhere herein. The methods may comprise administering said test molecule to a subject or patient prior to obtaining a sample from said subject or patient, wherein the obtained sample is the nonpurified test sample of use in the method. Thus, preferably the non-purified test sample is a sample which has been obtained from a subject or patient to whom a test molecule has been administered. Alternatively viewed, the non-purified sample on which the steps of the method are performed comprises said test molecule. Again, the control level is that determined (previously, or as part of the method) in a control sample that has not been exposed to said test condition, e.g. which has not been exposed to said test molecule (e.g. drug). The step of heating the non-purified test sample, and the subsequent separation and determination steps, may be performed at any time point after exposure of the non-purified test sample to the test condition (e.g. at any time point after drug treatment). In other words, the method of the invention may be performed at any time point after exposure of the non-purified test sample to the test condition (e.g. at any time point after drug treatment). Preferably, the method is performed within 24 hours, more preferably within 18 hours, more preferably within 12 hours, more preferably within 8 hours of exposure of the non-purified test sample to the test condition. Cleavage may be initiated within 1 hours of exposure to the test condition, and may advantageously be detected within an 8 hour timeframe, e.g. after 1 hour, 2 hours, 4 hours, 6 hours and / or 8 hours of exposing the non-purified test sample to the test condition. Again, these time frames are preferably after drug administration. Typically, where the test molecule is present extracellularly e.g. in solution, this may be simply added to the non-purified sample. Alternatively, the test molecule may be expressed recombinantly from a vector encoding the test molecule. The step of adding the test molecule may therefore involve transforming or transfecting a cellular sample with the vector encoding the test molecule and / or inducing expression of the test molecule from the vector in a cellular sample once transformation or transfection has been carried out. The step of adding the test molecule further includes inducing expression of a test molecule encoded by a gene naturally occurring in a cellular sample. Further, the method may require an extracellular test molecule to be transported into the cell. Alternatively, or additionally, a step of lysis may be carried out before, simultaneously or after the test molecule has been added. Such a lysis step allows contact between the proteins in the cells in the sample and the test molecule. Thus, any necessary lysis step is generally carried out before the separation step of the method of the invention. The methods of the invention may comprise as step of repeating the earlier steps with one or more (e.g. 2 or more, preferably 3 or 4 or more) different concentrations of the test molecule. As mentioned above, the level of each signature peptide that is determined is the level of the signature peptide relative to a control level of the same signature peptide. The methods of the invention may comprise performing the indicated steps on the test sample, and on a control sample, in order to determine the levels of signature peptides in each. If the method of the invention comprises repeating the steps of the method on a non-purified control sample, then said repeated steps can be performed simultaneously to, before or after the steps performed with the non-purified test sample. The test and control samples may therefore be obtained and treated at different times, and simultaneous or directly sequential processing is not necessary (although can be done). The period of time between performing the steps on the test and control samples may be any period of time, e.g. a few minutes (e.g. between 1-10 minutes), hours (e.g. from 1, 2, 5, 10 hours), months (from 1, 2, 5, 10 months) or years (from 1, 2, 3, 4 or 5 years). Particularly, it will be appreciated that in the case where the samples are obtained from the same patient, e.g. at different time points, a significant period of time may occur between collection if the samples and / or performance of the method thereon. However, performance of the method steps on a control sample is not essential for the performance of the method. Rather, the level of each signature peptide determined as part of the method of the invention may be compared to a control level that has been obtained previously, e.g. via a “reference database” or “control database”. In other words, the data obtained with the control sample may be in the form of reference data, e.g. a reference database, and the methods of the invention need not comprise the performance of steps on a control sample. As mentioned above, in all aspects and embodiments, preferably step c) comprises subjecting either or both the soluble and insoluble protein fractions of step b) to mass spectrometry analysis to produce mass spectrometry data; and analysing the mass spectrometry data to determine said levels of signature peptides. Preferably the control levels are also determined using mass spectrometry. In all cases, preferably step c) is performed using the soluble protein fraction produced in step b). As explained above, the methods of the invention preferably comprise heating the nonpurified sample to a series of temperatures in a temperature range to obtain a plurality of heated test sample aliquots, which are then each processed by the subsequent steps of any method herein. Thus, it follows, that if a plurality of heated test sample aliquots are obtained then the methods preferably comprise determining the relative level of the signature peptide(s) in each heated test sample aliquot as compared to the level of the same signature peptide(s) in the equivalent aliquot of a suitable control sample heated to the same temperature point. It is then possible to prepare a plot of the first and second relative levels discussed above (e.g. as fold changes) at each temperature point. Cleavage of the protein of interest is determined by a difference between said first and second relative levels determined using any one or more the heated sample aliquots, i.e. at any one or more temperature points. In methods such as this in which a plurality of heated test sample aliquots are obtained and processed, it is of course the case comparison to a control level would be performed for each heated test sample aliquot by comparison to an equivalent (or corresponding) heated control sample aliquot (i.e. which had been heated to the same temperature point in the series). The combined relative levels of a plurality of signature peptides of a first domain can be determined at each temperature point (i.e. in each heated test sample aliquot), and the combined relative levels of a plurality of signature peptides of a second domain can be determined at each temperature point (i.e. in each heated test sample aliquot). Such data is shown for instance in Figure 1. Cleavage is detected by a difference between the combined relative level of peptides the first domain and the combined relative level of peptides of the second domain, at at least one temperature point (i.e. in a protein fraction of at least one heated test sample aliquot). The relative levels of a given signature peptide at two or more, preferably all, of the temperature points used in the method can be combined (e.g. summed) to provide a single temperature-combined relative level, e.g. a “temperature-summed relative level”, which again may be expressed in terms of a fold change (i.e. a “temperature-summed fold change”). This is shown for instance in Figure 2, panel (ii) for a number of different signature peptides in two different domains of a protein of interest. Thus, optionally, the relative levels of a signature peptide determined using each heated test sample aliquot (i.e. the relative levels at each temperature) are summed to provide a “temperature-summed relative level” for that signature peptide. This may be performed for the at least two signature peptides, preferably each signature peptide analysed. These temperaturesummed relative levels are of subsequent use in further analysis. However, they may also be used to determine cleavage of a protein of interest, since cleavage of the protein of interest is determined by a difference between a temperature-summed relative level of one or more signature peptides of the first domain and a temperature-summed relative level of one or more signature peptides of the second domain, e.g. by a difference between the temperature-summed relative levels of said first and second signature peptides. Mass spectrometry is the preferred means for determining the levels of the signature peptides in the protein fraction(s) of the test sample (or aliquots), and in the protein fractions(s) of the control sample (or aliquots), which then permits calculation of the above-mentioned relative levels and temperature-summed relative levels for signature peptide(s) of interest. The signature peptides identified in a protein fraction may be labelled and ordered relative to the protein of interest on a sequence basis, and plotted sequentially (in the N- to C-terminus direction or vice versa) on the x-axis of a plot in which the y-axis represents the abovedescribed relative levels (at any given temperature) or temperature-summed relative levels (e.g. temperature-summed fold change) for each peptide. A cleavage site may then be located in the protein of interest as existing between the two adjacent signature peptides between which there is the largest difference in the determined relative levels or temperature-summed relative levels. If desired, the cumulative sum of the temperature-summed relative levels (e.g. temperature-summed fold-changes) determined as described above for each signature peptide identified in a protein fraction can be calculated and plotted, being ordered according the signature peptide’s position in the protein on the x axis (see for instance Figure 2, panel (iii)). In other words, the signature peptides identified in the protein fraction may be ordered relative to the protein of interest on a sequence basis and plotted sequentially (in the N- to C-terminus direction or vice versa) on the x-axis of a plot in which the y-axis represents the cumulative sum of the above-described temperature-summed relative levels (e.g. summed fold change) for each peptide. Thus, such a plot may show the cumulative temperature-summed fold changes of all detected signature peptides for a protein of interest (or all signature peptides from the first and second domain), reading in the N to C direction on the x-axis. An example of such a plot is shown in Figure 2, panels (iii) and (iv). These plots may also be used to determine cleavage of a protein of interest, since cleavage of the protein of interest is determined by the presence of an inflexion point in the plotted curve. A linear relationship between the cumulative temperature-summed relative levels and the protein’s signature peptide sequence order is indicative of every peptide having a similar relative level vs. control, and so is indicative that the protein is not cleaved. If the relationship between the cumulative temperature-summed relative levels and the protein’s signature peptide sequence order is non-linear, i.e. if there is an inflexion point, then it is indicative of peptides to one side of the inflexion point having a different relative level (vs. control) as compared to the peptides on the other side of the inflexion point (vs. control), i.e. this is indicative of the protein of interest being cleaved in the test sample. If desired, the position of the inflexion point on the curve relative to the x-axis can be used to determine the location of the cleavage site in the protein of interest. If the curve is concave, the minimum of the second derivative of the curve is the inflexion point and if it is convex, then the maximum of the second derivative of the curve is the inflexion point. The inflexion point may be determined computationally. Thus, the methods of the present invention preferably may further comprise locating a cleavage site in the protein of interest by: ordering the signature peptides that are present in the protein fraction of said heated test sample or aliquot thereof on the basis of their sequence alignment with the protein of interest, in either the N-terminus to C-terminus direction or the C-terminus to N-terminus direction, wherein a cleavage site is located in the protein of interest between the two adjacent signature peptides between which there is the largest difference in the determined relative levels or temperature-summed relative levels; or wherein a cleavage site is located by the presence of an inflexion point between two adjacent signature peptides in a plot of cumulative temperature-summed relative levels. Thus, the present invention also provides a method for determining the cleavage site of a cleaved protein of interest in a non-purified sample, wherein said protein of interest comprises a plurality of signature peptides in said first domain, and a plurality of signature peptides in said second domain, and wherein the method comprises the steps of: a) heating the non-purified test sample, wherein said heating step comprises i) heating the non-purified test sample to a series of temperatures in a temperature range and obtaining an aliquot of the sample after heating at each temperature, or ii) obtaining a plurality of non-purified test sample aliquots and heating each sample aliquot to a different temperature within a series of temperatures in a temperature range, to obtain a plurality of heated test sample aliquots; b) separating soluble from insoluble protein in each of the heated test sample aliquots to produce soluble and insoluble protein fractions thereof; and c) using either the soluble or insoluble protein fractions i) determining the relative level of each signature peptide in the protein fraction of each heated test sample aliquot, as compared to a control level of said signature peptide, ii) calculating a temperature-summed relative level for each signature peptide, which is the sum of the relative levels of the signature peptide determined in the protein fraction of each heated test sample aliquot; and d) ordering said temperature-summed relative levels of the signature peptides on the basis of the sequence alignment of the signature peptides with the protein of interest, in either the N-terminus to C-terminus direction or the C-terminus to N-terminus direction, wherein a cleavage site is determined in the protein of interest at the position between the two adjacent signature peptides between which there is the largest difference in the determined temperature-summed relative levels. Alternatively step d) comprises d) ordering the cumulative sum of said temperature-summed relative levels of the signature peptides on the basis of the the sequence alignment of the signature peptides with the protein of interest, in either the N-terminus to C-terminus direction or the C-terminus to N-terminus direction, wherein a cleavage site is determined in the protein of interest at the position between the two adjacent signature peptides between which there is an inflexion point. The present invention also provides a method for identifying a cleaved protein within a non-purified sample, said method comprising performing the steps of a method of the invention as defined above, and where cleavage is detected, subsequently determining the sequence of at least one of said signature peptides, and using said sequence of at least one of said signature peptides to search a protein sequence database in order to identify the protein of interest from which the signature peptide(s) is / are derived. The above described methods comprise a step of separating soluble and insoluble proteins and domains after heating the non-purified test sample (step b). This is preferred, however, such a separation step is not required. As explained above, after heating, the heated test sample (or heated test sample aliquots) will comprise a mixture of correctly folded (soluble) and misfolded (insoluble) proteins and domains. The levels of the soluble protein domains can be determined in the heated sample (or heated aliquots) without separation of the sample into distinct soluble and insoluble protein fractions. For instance, many antibodies and affinity reagents that recognise the correctly folded structure (soluble) of a domain will bind the soluble domain with much higher affinity than the unfolded and precipitated domain. Antibodies and other affinity reagents can be linked to magnetic beads or column resin which is mixed with the heat treated non-purified sample (or aliquot). This mix can in a subsequent step be put in an appropriate valve and washed to remove insoluble domain when this does not have high affinity to the affinity reagent. The amount of domain bound to the affinity reagent can subsequently be measured using for example Bradford techniques, gel electrophoresis, Elisa or surface plasmon resonance detection. It is also possible to use homogenous immune assays where the soluble domain is detected by at least two different antibodies, such as alpha-screens, Elisa assays or proximity ligation assays (PLA, Blokzijl, J Intern Med 2010 268;232). When the right antibody combination has been established which specifically recognises the soluble domain, this may give a direct signal for the amount of soluble domain present in the sample, while precipitated domain does not provide a signal. When such separation of the signal from the soluble domain and the insoluble (precipitated) domain can be directly accomplished in a homogenous assay, no physical separation of the soluble and insoluble (precipitated) domains is required to determine the level of the soluble domain. Thus, when the method of detection involves the use of one, or two different, affinity reagent(s), a heated sample separation step may be excluded. A separation step may however still be performed, with detection of the soluble domain being made in the soluble protein fraction that is produced. Thus, the invention provides a method for detecting cleavage of a protein of interest within in a non-purified test sample, wherein said protein of interest comprises at least a first signature peptide present in a first domain and at least a second signature peptide present in a second domain, wherein the method comprises the steps of: d) heating the non-purified test sample to provide a heated test sample; e) optionally separating soluble from insoluble protein in the heated test sample of step a) to produce soluble and insoluble protein fractions of said heated test sample; and f) determining, in said heated test sample, or in the soluble and / or insoluble protein fraction thereof, i) a first relative level, which is the level of the first domain as compared to a control level, and ii) a second relative level, which is the level of the second domain as compared to a control level, wherein cleavage is detected by a difference between said first and second relative levels in said heated test sample or in the soluble and / or insoluble protein fraction thereof. The above described preferred and optional features, steps and embodiments described above in relation to the methods of the invention comprising a separation step apply mutatis mutandis to the methods of the invention that need not comprise a separation step. Thus, for the avoidance and doubt, in these alternative methods, the preferred and optional features, steps and embodiments of the heating step, the separation step (if performed), the protein of interest, the domains, the meaning of “cleavage”, the cleavage site, the non-purified test sample and the cell(s) therein, the plurality of heated test sample aliquots, the protein fractions, the optional lysis step, the meaning of a “level” and a “relative level”, the step of determining a level (i.e. a relative level), the means for measuring levels, optional sample processing steps, the “control”, control level, the control sample, etc. described anywhere else herein apply mutatis mutandis to these methods of the invention. Of course, these methods comprise the determinations of levels of domains rather than signature peptides thereof, so references made elsewhere herein to “signature peptides” are applicable to the domains of the present methods, unless clear from technical context that this is not the case. Preferably, the method comprises a) heating the non-purified test sample, wherein said heating step comprises i) heating the non-purified test sample to a series of temperatures in a temperature range and obtaining an aliquot of the sample after heating at each temperature, or ii) obtaining a plurality of non-purified test sample aliquots and heating each sample aliquot to a different temperature within a series of temperatures in a temperature range, to obtain a plurality of heated test sample aliquots; b) optionally separating soluble from insoluble protein in each of the heated test sample aliquots to produce soluble and insoluble protein fractions thereof; and c) determining, in each heated test sample aliquot, or in the soluble and / or insoluble protein fraction thereof, i) a first relative level, which is the level of the first domain as compared to a control level, and ii) a second relative level, which is the level of the second domain as compared to a control level, wherein cleavage is detected by a difference between said first and second relative levels in the heated test sample, or soluble and / or insoluble protein fraction thereof, or in at least one heated test sample aliquot or soluble and / or insoluble protein fraction thereof. Preferably step c) further comprises calculating a temperature-summed relative level of the first domain, which is the sum of the first relative levels determined in each heated test sample aliquot, or in each soluble or insoluble protein fraction thereof, and calculating a temperature-summed relative level of the second domain, which is the sum of the second relative levels determined in each heated test sample aliquot, or in each soluble or insoluble protein fraction thereof, wherein cleavage is detected by a difference between the temperature-summed relative levels of the first and second domains. Optionally, the plurality of heated sample aliquots obtained may be pooled to form a pool of heated sample aliquots, and said pool of heated sample aliquots is thereby the product of the heating step that is processed as set out in the subsequent steps of the method, i.e. the level of the domains may be determined in a pool of said heated sample aliquots. Clearly, determining the relative level of a domain as compared to a control level comprises determining the level in the heated test sample, or in the soluble / insoluble protein fraction thereof, before comparing the determined level to a control level. In these methods of the invention, the levels of the domains are determined in the heated test samples (or aliquots) or soluble protein fractions thereof. Determination of the level of a domain in a soluble or insoluble protein fraction may be performed using mass spectrometry, which is as described above. Mass spectrometric determination of levels of signature peptides of a domain permits the determination of levels of the domain itself. However, the level of a domain in a soluble or insoluble protein fraction may be determined by affinity binding. Determination of the level of a domain by affinity binding refers to the level of a domain being determined by affinity binding between the domain and an affinity reagent, for example an antibody, antibody fragment or affibody (non-Ab based protein binding partner). Preferably, antibodies, monoclonal or polyclonal, against the domain may be used. Antibody-based methods allow for rapid and reliable analysis of a wide variety of proteins, including those which themselves possess no catalytic activity. Such methods are routine in the field. It is within the competencies of the person of ordinary skill in the art to generate, select and / or obtain one or more affinity reagents against a desired domain, and to use the same in order to detect the level thereof in a sample or protein fraction. Affinity reagents are discussed below in the context of two such agents being used to detect a soluble domain in a heated sample without performance of a separation step, and the same preferred and optional features of such affinity reagents and their use apply mutatis mutandis to the affinity reagents and their use when applied to a soluble or insoluble protein fraction. If the soluble and insoluble protein fractions have been separated, the affinity reagent used to detect the domain in either of said fractions need not be specific for the soluble or insoluble form of the domain, it is sufficient for it to be specific for the domain per se. In general, methods exploiting affinity binding to detect specific proteins or domains rely on the coupling of the affinity regent to an enzyme, fluorophore or DNA to allow quantification of the amount of the protein / domain of interest. Alternatively, the detection reagent can be coupled to a secondary affinity reagent that binds to the protein- or domain-specific affinity reagent. Plate based assay formats where the affinity reagent is coupled to an enzyme (enzyme-linked immunosorbent assay (ELISA)) can be used. Homogenous assays, such as AlphaLISA and PEA, in which two affinity reagents binding the same protein / domain region are required, are useful since this increases the specificity and eliminates the need of washing steps. Imaging formats and FACS based techniques have the advantage of allowing analysis of protein / domain levels in individual cells. Determination of the level of a domain by affinity binding may of course be used if the identity (amino acid sequence) of the domain is known. However, even if the identity of the domain(s) is / are unknown, affinity based methods may still be used. For instance, antibody arrays may be used to determine the level of multiple domains / proteins in a sample simultaneously; such methods allow for rapid and reliable analysis. Protein affinity arrays, based on specific antibodies, can be used to quantify proteins / domains. Preferably, the step of determining the level of a first or second domain in the heated test sample or aliquot, or soluble protein fraction thereof, is achieved by using at least two affinity reagents specific for the soluble form of the first or second domain, as appropriate. The affinity reagents are preferably antibodies. However, it will be appreciated that in this aspect of the invention other affinity reagents may be capable of detecting a soluble domain in a sample in a similar way to the two antibodies described above. In this aspect, a separation step is unnecessary as the method comprises determination of the level of soluble (or native) domain using two or more affinity reagents (preferably antibodies) that are specific for the soluble form of the domain. By specific is meant that the affinity reagents bind with a higher affinity to the soluble or native form of the domain than to the unfolded or insoluble (precipitated) form of the domain. In this way, such affinity reagents can determine whether soluble or native forms of the domain are present in a sample after heating. The two or more affinity reagents must together be capable of distinguishing between soluble or native, and unfolded and / or insoluble forms of a domain and thus must be capable of detecting soluble or native domain against a background of other proteins (both soluble and insoluble) and any insoluble domain that may also be present. This form of the method of the invention is particularly advantageous as there is no specific requirement to carry out a separation step, and particularly no separation step may be carried out. In this respect, the method involves minimal processing steps, which potentially allows automation of the method and an increased throughput, where large numbers of samples can be handled. The “affinity reagent” as used herein thus refers to any reagent which is capable of binding with a higher affinity to a soluble or native form of a domain / than to an unfolded and / or insoluble form of the same domain. An affinity reagent which binds with a higher affinity to a soluble or native form of a domain compared to the unfolded and / or insoluble form of the domain will have a smaller Kd value for its association with the soluble or native target domain than for its association with the unfolded and / or insoluble form of the domain. Particularly, an affinity reagent of the present application may have a Kd value which is at least 100 times smaller with respect to binding to the soluble or native form of the domain than the Kd with respect to binding to the unfolded and / or insoluble form of the domain. Methods for measuring the Kd values of affinity reagents are well known in the art. Thus, the use of two or more affinity reagents (e.g. antibodies) for the detection of the soluble or native domain allows the use of affinity reagents which may have a lower specificity for the soluble or native domain than if a single affinity reagent (e.g. antibody) was used alone to detect the soluble or native domain. As two or more affinity reagents (e.g. antibodies) are required to be bound to the soluble or native domain to result in its detection, less specific affinity reagents can be used and can still result in a specific method of detecting the soluble or native domain. Thus some binding of at least one of the affinity reagents (e.g. antibodies) may occur to the unfolded and / or insoluble form of the domain, although binding to the soluble or native form of the domain is preferential and association may be at least 100 times greater than association to the unfolded and / or insoluble form of the domain. Similarly, each affinity reagent particularly binds with higher affinity to the soluble or native form of the domain than to any other domain / protein present in the sample. Preferably, at least one, preferably at least two, of the affinity reagents (e.g. antibodies) used in the method is capable of binding specifically to the soluble or native form of the domain but not to the unfolded and / or insoluble form of the domain (or to any other protein). Thus, the reagent(s) may bind specifically to the soluble or native domain and any binding to the unfolded and / or insoluble domain may be non-specific and minimal. Preferably, at least one affinity reagent specific for the domain is capable of binding only to the soluble or native domain and not to the unfolded or insoluble domain, while one or more other affinity reagents specific for the domain may be capable of binding to both forms, but binding to the soluble or native form of the domain is with higher affinity than binding to the unfolded and / or insoluble form of the domain. Further, the method provides for the use of two or more affinity reagents which bind specifically to the soluble or native form of the domain but not to the unfolded and / or insoluble form of the domain. In order to distinguish soluble (or native) from unfolded and / or insoluble domain, the affinity reagent may recognize (particularly specifically recognize) an epitope or sequence of the domain which is exposed in the soluble or native form of the domain but not in the unfolded and / or insoluble form of the domain. The two or more affinity reagents recognise different epitopes or sequences on the domain and thus provide a more specific method of distinguishing soluble or native from unfolded and / or insoluble domain, than when identifying a single epitope or sequence. (Although with proteins which are homodimers, the affinity reagents may be directed to the same epitope). Thus, both (or more) affinity reagents must be bound to the domain in order to determine that a soluble or native form of the domain is present. A positive detection of soluble or native domain is only achieved if both (or more) affinity reagents (e.g. antibodies) are bound. The affinity reagent may be an antibody, antibody fragment, affibody, peptide, aptamer, DARTs or other small molecule that binds to the soluble or native form of a domain with a higher affinity than to the unfolded and / or insoluble form of the domain. Particularly, at least one affinity reagent is an antibody and more particularly two antibodies are used per domain to be detected in the method. The invention however also encompasses the use of different affinity reagents e.g. the use of an antibody and another affinity reagent. Since more than one domain may be analysed in each sample, multiple affinity reagents may be used in each analysis step, where at least 2 affinity reagents are used for each domain to be detected. Where multiple domains are to be analysed, the signals generated by the binding of the two or more affinity reagents to each domain should be different. The detection of the two or more affinity reagents (particularly two antibodies) may be using a reporter assay which results in a signal change when the two or more affinity reagents are bound to soluble or native domain. Generally, the two or more affinity reagents are labeled (particularly with different labels) and the close proximity of those labels to one another when bound via the affinity reagents to the soluble or native form of the domain results in a change in signal e.g. the emission of fluorescence or the production of light or fluorescence at a different wavelength (to the labels when used alone) or the quenching of fluorescence. Such reporter assays are often referred to as proximity reporter assays, e.g. a FRET (fluorescence resonance energy transfer) based method (or a variant thereof, such as BRET (Bioluminescence Resonance Energy Transfer)) may be used in the present method for detection, where the close association of one label (a donor molecule) attached to an affinity reagent, to a second label (an acceptor molecule) attached to an affinity reagent, results in the production or alteration of a signal. Thus, the presence of the two or more labels bound to the soluble or native domain via the affinity reagents results in a detectable signal change. For instance, FRET, transfer of energy from the donor to the acceptor molecule labels may result in the emission of fluorescence by the acceptor molecule. In this way, the labeling of two (or more) affinity reagents (antibodies) which bind to the soluble form of a domain, with labels which have a signal change when in close proximity to each other, can result in the emission of fluorescence when both are bound to the soluble domain, thus enabling detection of the soluble target domain. Donor and acceptor molecules used in FRET / BRET based methods are well known in the art and include pairs such as cyan fluorescent protein and yellow fluorescent protein (both variants of green fluorescent protein); and bioluminescent luciferase and YFP. Such a method requires the binding of both (or more) affinity reagents (e.g. antibodies) to the soluble domain before detection of any signal (e.g. luminescence) is achieved. In this aspect, one affinity reagent may be labeled with donor molecule and the second affinity reagent may be labeled with the acceptor molecule. The labels which demonstrate a signal change when in close proximity (e.g. when bound via affinity reagents to the soluble domain),e.g. donor and acceptor molecules, may be coated or comprised within separate bead populations which may then be used to bind to each affinity reagent (antibody). Thus, the beads coated with one of the labels (e.g. the donor molecule) may be used attached to the first affinity reagent e.g. antibody and the beads coated with the second label (e.g. the acceptor molecule) may be used to detect the second affinity reagent (e.g. antibody). Each bead population (e.g. donor or acceptor) may be conjugated with a further reagent to allow binding to either the first or second affinity reagent (e.g. antibodies). For example, a bead population (donor or acceptor) may be streptavidin coated to allow binding to biotinylated affinity reagent (e.g. antibody) or maybe conjugated to protein A to allow binding to an antibody affinity reagent. Methods of attaching beads to affinity reagents such as antibodies are well known in the art. It will be appreciated that the two or more affinity reagents (e.g. antibodies) may be labeled (e.g. with donor or acceptor molecules) prior to addition to the sample or after addition to the sample. However, particularly, the affinity reagents (antibodies) may be labeled before addition to the sample. The AlphaScreen Surefire assay format (Perkin Elmer) may be particularly used in the method of the invention, where other antibodies may be attached to the beads provided (see Osmond et al, Analytical biochemistry, 403, 94-9101, 2010, incorporated herein by reference). Other methods for detection of the bound affinity reagents include proximity ligation assays (such as Duolink from Olink) and ELISAs. Methods for producing affinity reagents, such as antibodies which may bind to the soluble form of a domain but not the insoluble form of a domain are known in the art. For example, the study of the 3D structure of a soluble and insoluble protein can enable determination of epitopes which are exposed on the soluble form but not the insoluble form. Antibodies or peptides which bind to such epitopes can then be produced using standard methods. Further, methods are known which can be used to identify antibody pairs that bind to a domain e.g. methods employing surface plasmon resonace biosensors or ELISA. Further, Bembenek et al (Analytical Biochemistry, 408, 2011, 321-327, incorporated herein by reference), reported a bead based screening method using antibody capture on Protein A Alphascreen beads to analyse and select pairs of antibodies capable of binding to the same target. Thus, according to the methods of the invention it is possible to determine the level of the soluble first and second domains in the whole heated sample (the product of the heating step), i.e. a step of separating soluble and insoluble protein fractions is not necessary. A control level of a domain is the level determined in a corresponding (or equivalent) heated control sample (or aliquot) or soluble fraction thereof. The control sample is a non- purified control sample. The control level is determined in a heated sample (or aliquot) or soluble protein fraction thereof using the same (corresponding) method steps as used to determine the level in the test sample. The person of ordinary skill in the art understands that comparison to a control level means comparison of the level of same domain in the same sample type or protein fraction type. Thus, the level of a domain in the heated test sample (or aliquot) would be compared to the level of the same domain in the heated control sample (or aliquot). The level of a domain in a soluble protein fraction of the heated test sample (or aliquot) would be compared to the level of the same domain in the soluble protein fraction of the heated control sample (or aliquot). Preferably the control, i.e. the control sample / aliquot / fraction, referred to for determining the relative level of a first domain is the same control, i.e. the same control sample / aliquot / fraction, referred to for determining the relative level of the second domain. The present inventors have determined that detection of cleavage of one or more of certain proteins via the above-mentioned CETSA-based methods is indicative of apoptosis in a cell. The present invention therefore also provides the following methods. The preferred and optional features and embodiments described above, i.e. in relation to the methods for detecting cleavage of the invention, apply mutatis mutandis to each and all of the further methods described below. In a further aspect the present invention provides a method of detecting apoptosis in a cell in a non-purified test sample by detecting cleavage of a protein of interest within or on said cell, wherein the protein of interest is selected from the group consisting of PARP1, DDX21, LMNB2, LMNB1, BCAP31 and MATR3; and wherein said method comprises the steps of any of the methods of the invention described elsewhere herein, wherein detection of cleavage of at least one of said proteins of interest is indicative of apoptosis. As used herein, “apoptosis” refers to cell death by an intracellular controlled process. Apoptosis is characterized by a condensation and subsequent fragmentation of the cell nucleus, and systematic degradation of cellular components, during which the plasma membrane remains intact. The apoptosis detected according to the present invention may be any category or type of apoptosis, including but not limited to extrinsic apoptosis, intrinsic apoptosis, caspase-mediated apoptosis, caspase-independent apoptosis, tumour necrosis factor (TNF)-related apoptosis, FAS ligand-mediated apoptosis, nuclear apoptosis, early nuclear apoptosis, drug-induced apoptosis, pathological apoptosis, aberrant apoptosis, and so on. The apoptosis detected is preferably that induced by a test condition (discussed elsewhere herein) to which the test sample has been (or is) exposed. For instance, the apoptosis detected may be that induced by a test molecule (discussed elsewhere herein, e.g. a drug) to which the test sample has been (or is) exposed. Preferably the apoptosis is nuclear apoptosis (which may be drug-induced), more preferably early nuclear apoptosis (which may be drug-induced). As is clear, the apoptosis detected or investigated according to the present invention is apoptosis of the cells in the non-purified test sample. The methods of the invention relating to apoptosis detection may comprise detecting the levels of signature peptides and / or domains from one or more, preferably at least two, preferably at least three, preferably each of said proteins of interest, in order to assess for cleavage thereof. Preferably, apoptosis is indicated by detection of cleavage of at least two, more preferably at least three of the proteins of interest selected from the group consisting of PARP1, DDX21, LMNB2, LMNB1, BCAP31 and MATR3. Preferably the protein of interest is PARP1. Preferably, the methods of the invention comprise detecting the levels of signature peptides and / or domains from PARP1, and preferably one or more, preferably two or more further of said proteins of interest, in order to assess for cleavage Preferably apoptosis is indicated by detection of cleavage of PARP1, more preferably PARP1 and any one or more, preferably two or more, further of said proteins of interest. In all cases, the detection of cleavage of further proteins may also be performed as part of the method. The proteins of interest named herein are human proteins, but the protein of interest detected may be homologous proteins from other species. In all cases, the protein of interest is preferably not directly bound by any test molecule (e.g. drug) that is applied to the sample, or administered to the subject from which the sample is obtained. Thus preferably the protein of interest is not a target protein for a drug that is applied to the sample, or administered to the subject from which the sample is obtained. In such methods of detecting apoptosis, cleavage of the protein(s) of interest may be detected in the heated sample (or aliquot), or in the soluble and / or insoluble protein fraction thereof, as described above. As described above, the control level may be that determined (previously, or as part of the method) in a control sample that has not been exposed to a test condition to which the test sample has been exposed. For instance, the test sample may have been exposed to a test molecule, e.g. a drug, which the control sample has not been exposed to. The step of heating the non-purified test sample, and the subsequent separation and determination steps, may be performed at any time point after exposure of the non-purified test sample to the test condition (e.g. at any time point after drug treatment). In other words, the method of the invention may be performed at any time point after exposure of the non-purified test sample to the test condition (e.g. at any time point after drug treatment). Preferably, the method is performed within 24 hours, more preferably within 18 hours, more preferably within 12 hours, more preferably within 8 hours of exposure of the non-purified test sample to the test condition. Cleavage may be initiated within 1 hours of exposure to the test condition, and may advantageously be detected within an 8 hour timeframe, e.g. after 1 hour, 2 hours, 4 hours, 6 hours and / or 8 hours of exposing the non-purified test sample to the test condition. Again, these time frames are preferably after drug administration. Detection of protein cleavage, and so apoptosis, at these time points is advantageous, since there remains a need in the field for methods capable of early detection of apoptosis. Thus, the methods of the invention may also be considered as methods of detecting the initiation of apoptosis. Preferably, when detecting the initiation of apoptosis, the method is performed within the above-discussed periods following exposure of the non-purified test sample to the test condition. Alternatively viewed, the invention provides a method of detecting the initiation of apoptosis in a cell in a non-purified test sample, wherein the protein of interest is selected from the group consisting of PARP1, DDX21, LMNB2, LMNB1, BCAP31 and MATR3; and wherein said method comprises the steps of any of the methods of the invention described elsewhere herein, wherein detection of cleavage of at least one of said proteins of interest is indicative of apoptosis. The methods of the present invention are useful for the identification of compounds that inhibit or stimulate apoptosis. Such compounds may be useful therapeutics, e.g. in neurodegenerative disease and oncology therapies. Thus, in a further aspect, there is provided a method for identifying an apoptosis-inducing agent by detecting cleavage of a protein of interest within or on a cell in a non-purified test sample, wherein the non-purified test sample has been exposed to a test compound, wherein the protein of interest is selected from the group consisting of PARP1, DDX21, LMNB2, LMNB1, BCAP31 and MATR3; and wherein said method comprises the steps of any of the methods of the invention described elsewhere herein, wherein detection of cleavage of at least one of said proteins of interest is indicative of the test compound being an apoptosis inducing agent. In this aspect, the control sample has not been exposed to said test compound, or has been exposed to a lower amount (concentration) of said test compound. These methods can be alternatively viewed as methods of assessing the ability of a test compound to induce or enhance apoptosis of cells in a non-purified test sample In a related further aspect, there is provided a method for identifying an apoptosisinhibiting agent by detecting inhibition of cleavage of a protein of interest within or on a cell in a non-purified test sample, wherein the non-purified test sample has been exposed to a test compound and an apoptosis-inducing agent, wherein the protein of interest is selected from the group consisting of PARP1, DDX21, LMNB2, LMNB1, BCAP31 and MATR3; and wherein said method comprises the steps of any of the methods of the invention described elsewhere herein, wherein the test compound is indicated as an apoptosis inducing agent when cleavage of at least one of said proteins of interest is detected at a lower level than in a control sample exposed to said apoptosis-inducing agent but not to said test compound. Apoptosis inducing agents are well-known in the field, and any such agent may be used, e.g. venetoclax. The level of cleavage determined may correlate with the number of the proteins of interest for which cleavage is detected (a higher number being cleaved indicating a higher level of apoptosis), or by the extent (magnitude) of the differences between the relative level(s) of signature peptides / domains determined as part of the method. The extent of the difference may be determined on an individual protein of interest basis, or on the basis of the sum of the differences in the levels determined for a plurality of the proteins of interest. These methods can be alternatively viewed as methods of assessing the ability of a test compound to inhibit apoptosis of cells in a non-purified test sample. These methods of identifying apoptosis inducing or inhibiting agents may comprise a step before the heating step of exposing the non-purified test sample to said test compound. Preferably the compound is a test compound as defined elsewhere herein. Preferably the compound is a drug, e.g. an anticancer agent. Preferably the method is a high-throughput (drug) screen. Also provided is a method of monitoring the effectiveness of a therapeutic treatment by detecting cleavage of a protein of interest within or on a cell in a non-purified test sample obtained from a subject, wherein said subject has received said therapeutic treatment, wherein the protein of interest is selected from the group consisting of PARP1, DDX21, LMNB2, LMNB1, BCAP31 and MATR3; and wherein said method comprises the steps of any of the methods of the invention described elsewhere herein, wherein said control level is determined in a control sample obtained from said subject at an earlier time point in the course of therapeutic treatment. The level of cleavage detected is indicative of the effectiveness of the treatment. Preferably the sample is a patient sample, preferably a cancer patient wherein said cells in said non-purified sample are cancer cells. The sample may be a tumour sample, e.g. biopsy. Preferably the therapeutic treatment is a chemotherapy or radiotherapy treatment. An increase in the level of cleavage detected indicates increased apoptosis, and so an increased effectiveness of treatment, conversely a decrease in the level of cleavage indicates decreased apoptosis and a decreased effectiveness of treatment. As mentioned above, the level of cleavage determined may correlate with the number of the proteins of interest for which cleavage is detected (a higher number being cleaved indicating a higher level of apoptosis), or by the extent (magnitude) of the differences between the relative level(s) of signature peptides / domains determined as part of the method. The extent of the difference may be determined on an individual protein of interest basis, or on the basis of the sum of the differences in the levels determined for a plurality of the proteins of interest. Chemotherapy and radiotherapy treatment often induce high levels of apoptosis. However, neoplastic cells may be resistant to treatment. For example, in the case of leukaemia, particularly acute leukaemias, failure of malignant cells to undergo cell death in response to chemotherapy, is a major cause of treatment failure. As such, methods that are capable of detecting drug or radiotherapy-resistant cells, e.g. cancer cells, as well as a means to evaluate treatment effectiveness, would be desirable. The present invention provides such methods. Thus, in a further aspect, the present invention provides a method of identifying cells resistant to a therapeutic treatment by detecting inhibition of cleavage of a protein of interest within or on said cells in a non-purified test sample, wherein the non-purified test sample has been exposed to a therapeutic treatment, wherein the protein of interest is selected from the group consisting of PARP1, DDX21, LMNB2, LMNB1, BCAP31 and MATR3; and wherein said method comprises the steps of any of the methods of the invention described elsewhere herein, wherein resistance to said therapeutic treatment is identified when cleavage of said proteins of interest is not detected, wherein the control sample has not been exposed to said therapeutic treatment, or cleavage of at least one of said proteins of interest is detected at a lower level than in a control sample comprising treatment responsive cells that has also been exposed to said therapeutic treatment. The level of cleavage determined may be as described above. Preferably the sample is a patient sample, preferably a cancer patient wherein said cells in said non-purified sample are cancer cells. The sample may be a tumour sample, e.g. biopsy. Preferably the therapeutic treatment is a chemotherapy or radiotherapy treatment. These methods may comprise a step before the heating step of exposing the non-purified test sample (and in embodiments, the control sample) to said treatment, or administering said treatment to said patient from which the patient sample has been obtained. Also provided is a method of diagnosing an apoptosis-associated condition or disease in a subject. Preferably the subject is a mammal, preferably a human. The method comprises performing a method of the invention for detecting cleavage of a protein of interest within or on a cell in a non-purified test sample, wherein the non-purified sample is a sample obtained from the subject, wherein the protein of interest is selected from the group consisting of PARP1, DDX21, LMNB2, LMNB1, BCAP31 and MATR3, and wherein the condition or disease is diagnosed by the detection of apoptosis. Apoptosis-associated conditions are those characterised by or caused by aberrant apoptosis, e.g. excessive apoptosis, and the disease may be selected from the group consisting of chronic neurodegenerative disease, cancer, sepsis, trauma, hypoxia, anoxia, ischemia, spinal trauma, head trauma, lesions (wounds), and toxinexposure. The invention will now be further described in the following non-limiting Examples in which: Figure 1 shows the relative level of signature peptides detected in the soluble protein fraction of heated aliquots of a venetoclax-treated (6h) test sample of Kasumi-1 cells, as compared to the level in heated aliquots of a non-treated control sample of Kasumi-1 cells. The four panels relate to four different proteins of interest. The relative levels of a plurality of signature peptides from a first domain of a given protein as compared to controls, and the relative levels of a plurality of signature peptides from a second domain of said protein as compared to controls, were determined. Peptide levels were determined by mass spectrometry. The relative levels of the signature peptides of the first domain (vs. control) were summed, as were the relative levels of the signature peptides of the second domain (vs. control). In each panel, the left-hand series of bars shows the summed relative levels for the signature peptides of the first domain at each temperature point, and the right-hand series of bars shows the summed relative levels for the signature peptides of the second domain at each temperature point. Summed relative levels are expressed as the log2 fold-change of the sum of all measured peptides in said domain as compared to in control sample. Aliquots of the samples were taken, and after heating each aliquot to a different temperature in a series of temperatures (37, 47, 50, 52, 54 and 57°C), soluble and insoluble protein fractions were separated by centrifugation, and the soluble fractions of each heated sample aliquot were prepared for, and analysed by, mass spectrometry. Mass-spectrometry was performed using isobaric-labeling and 3 biological replicated for each condition to minimize measurement errors. Figure 2 shows how the cleavage status of a protein of interest (Protein X) in a nonpurified sample may be determined, and further how to determine the location of the cleavage site. Non-purified test and control samples are heated to a series of temperatures in a temperature range, heated sample aliquots are separated into soluble and insoluble fractions, and the soluble fractions of aliquots obtained at each temperature point are prepared for, and analysed by, mass spectrometry. The mass spectrometry data is analysed and compared to determine, at each temperature point, the relative level of each soluble peptide in the test sample as compared to the level in the control sample. Relative levels are expressed as log2 fold-changes for each peptide at each temperature (Panel (i)). To determine which protein domains have concerted shifting peptides, the temperature-summed relative levels, i.e. the sum of all-fold changes, at all of the temperature points is then calculated for each peptide (panel (ii)) starting from the most N-terminal peptides continuing to the most C-terminal peptides. Optionally, the cumulative sum of the temperature-summed fold-changes obtained in the previous step are calculated and plotted, being ordered according the peptide position in the protein (N- to C-terminus direction) on the x axis (panel (iii)). From panels (i) and / or (ii) it is already clear that the protein is cleaved; signature peptides of the N-terminal region of the protein (peptide 1-8) show clear fold changes, whereas the levels of signature peptides of the C-teminal region (Peptide 9-12) do not deviate significantly from control (insignificant fold change observed). This indicates that some peptides are of a domain that is more thermally stable after heating, and other peptides are of a domain that is less thermally stable after heating, i.e. the domains must be physically separated in the non-purified test sample, i.e. the protein is cleaved. Cleavage of the protein is also shown by the presence of an inflexion point in panel (iii). As shown in panel (iv), the inflexion point may be computed to determine the cleavage site of the protein of interest; in the example given in Figure 2(iv), the inflexion point reveals that the protein of interest is cleaved between peptides 8 and 9. Figure 3 shows how cleavage (and the cleavage site) of a protein of interest may be determined visually from data such as that presented in panels (iii) and (iv) of Figure 2, i.e. plots of cumulative summed log2 fold-changes (test vs control sample) for each peptide plotted according to the peptide position in the protein on the x axis. Three characteristic results are shown. A) If a protein of interest in the sample is not cleaved, then the plot from the cumulative sum shows a linear relationship. B) A situation where the difference in the level of N-terminal peptides of a protein of interest in the soluble fraction of test vs. control samples is greater than the difference between the level of C-terminal peptides in the soluble fraction of test vs. control samples, which demonstrates that the protein has been cleaved. The fact that peptides from different regions (domains) of the protein display different thermal stability changes in test vs. control samples indicates that the two regions of the protein exist in separate polypeptide products, i.e. the protein is cleaved in the non-purified test sample (and not in the control sample). C) A situation where the difference in the level of C-terminal peptides of a protein of interest in the soluble fraction of test vs. control samples is greater than the difference between the level of N-terminal peptides in the soluble fraction of test vs. control samples, which demonstrates that the protein has been cleaved. Again, the fact that peptides from different regions (domains) of the protein display different thermal stability changes in test vs. control samples indicates that the two regions of the protein exist in separate polypeptide products, i.e. the protein is cleaved in the non-purified test sample (and not in the control sample). Figure 4 shows the relative level of signature peptides detected in the soluble protein fraction of heated aliquots of a venetoclax-treated test sample, as compared to the level in heated aliquots of a non-treated control sample. The six panels relate to six different proteins of interest. The relative levels of a plurality of signature peptides from a first domain of a given protein as compared to controls, and the relative levels of a plurality of signature peptides from a second domain of said protein as compared to controls, were determined. Peptide levels were determined by mass spectrometry. The relative levels of the signature peptides of the first domain (vs. control) were summed, as were the relative levels of the signature peptides of the second domain (vs. control). Data presented as summed fold change (log2) compared to a control sample (Vehicle (0)), (n=3) ±SEM. In each panel, the left-hand series of bars shows the summed relative levels for the signature peptides of the first domain at each temperature point, and the right-hand series of bars shows the summed relative levels for the signature peptides of the second domain at each temperature point. Known caspase cleavage sites were found in between shifting and non-shifting regions. Figure 5 shows determination of five cleaved proteins (PARP1, LMNB1, LMNB2, MATR3 and BCAP31) in non-purified test samples at 4-8h timepoints after administration of 7 different drugs. Relative levels of signature peptides within two different domains (N-terminal and C-terminal) of each protein were determined and pooled, as described for Figures 1 and 3. Levels of peptides measured within the soluble fraction,. Data presented as fold change (log2) compared to a control sample (Vehicle (0)), (n=3) ±SEM. For each protein, left-hand panel relates to signature peptides in an N-terminal domain, middle panel relates to signature peptides in a C-terminal domain, and right-hand panel shows the difference in summed relative levels between the signature peptides of the N- and C- domains. Within each panel, bars of a given shade represent test samples exposed to a particular drug. Each bar represents the summed relative level of signature peptides at a given heating point. Figure 6 shows that effector caspases 3 / 7 are not activated until 20 hours after paclitaxel treatment. Caspase 3 / 7 activity measured with an imaging assays with paclitaxel and the ER Ca2+ import activator CDN 1163 as an extra control. Figure 7 shows a Western blot of cleaved PARP1, LMNB1 and MATR3 after 6h venetoclax treatment and inhibition of cleavage by addition of the pan-caspase inhibitor Z-VAD-FMK. EXAMPLES Example 1 The cells used in this study (Kasumi-1 AML cells) over-express Bcl-2 family proteins, which are often overexpressed in cancer cells. Bcl-2 family proteins block the intrinsic apoptosis pathways by engaging pro-apoptotic BH3-only proteins. Apoptosis was induced in cells of a non-purified test sample by administering venetoclax; a Bcl-2 inhibitor currently in clinical use for e.g., several hematological cancers, which rapidly induces apoptosis by attenuating apoptosis blockades. In the present assays, cells are activated to enter apoptosis, but apoptosis is blocked by specific anti / apoptotic proteins. Releasing this block via drug administration thereby rapidly induces apoptosis responses. CETSA experiments were performed using non-purified test (drug treated) and control (non-treated) Kasumi-1 AML cells. Drug treatment comprised administering 100 nM venetoclax to cells at 6 h incubation time. Aliquots of the samples were taken, and after heating each aliquot to a different temperature in a series of temperatures, soluble and insoluble protein fractions were separated by centrifugation, and the soluble fractions of each heated sample aliquot were prepared for, and analysed by, mass spectrometry. The mass spectrometry data was analysed to determine the level of every signature peptide in the soluble fractions of the sample aliquots heated at each temperature point. For each signature peptide, at each temperature point, the relative level of the peptide in the drug-treated and heated test sample as compared to in the non-treated control sample was determined. The relative level was expressed as the log2 fold-change of each peptide in the test sample as compared to in the control sample. The results for 6 proteins of interest are shown in Figure 4. PARP1, MATR3, LMNB1, LMNB2, DDX21 and BCAP31. Surprisingly, when analyzing the MS data at the signature peptide level, rather than at the protein level, the present inventors determined that CETSA shifts (i.e. changes in the level in the soluble fraction of test samples vs. control samples) were observed only for some of the peptides, rather than all, peptides in these proteins. The CETSA shifts were observed for peptides derived from a specific region (domain) of each protein, whereas the peptides for which CETSA shifts were not observed were derived from a different region (domain) of the protein. The present inventors realized that surprisingly, the CETSA method was able to detect cleavage of proteins in the sample; the fact that peptides from one domain were present at a higher level (vs. control) in the soluble protein fractions from another domain (vs. control) indicated that the different domains of the protein in the sample had different thermal stabilities after heating, and so must be structurally separated (i.e. the protein was cleaved). For each of these six proteins, due to increased relative levels of signature peptides from one domain in the soluble fraction vs. control, as compared to the relative levels of signature peptides from a second domain in the soluble fraction vs. control, it can be concluded that one of the two domains is more stable (correctly folded) after heating than the other domain, i.e. the domains are independent, i.e. the protein is cleaved. As shown in Figure 4, the N-terminal domain of the proteins PARP1, LMNB1, LMNB2 and DDX21 is more thermally stable than the C-terminal domain, since the relative level of peptides from the N-terminal domain (vs. control) in the soluble protein fraction is higher than the relative level of peptides from the C-terminal domain (vs. control). In the proteins BCAP31 and MATR3, the C-terminal domain was shown to be more thermally stable than the N-terminal. In either scenario, the difference in the thermal stabilities of the domains, as indicated by the differences in the relative levels vs. control of the peptides from one domain as compared to the other, indicates that the protein is cleaved in the non-purified test sample. The present inventors term this phenomenon the REgional Stabilization after Proteolysis (RESP) effect. The RESP effects observed are consistent with known caspase cleavage sites for these proteins. Indeed, the inventors confirmed that PARP1, LMNB1 and MATR3 are cleaved after only 6 hour venetoclax treatment, and that this effect is caspase dependent (see Fig. 7, which shows a Western blot of cleaved PARP1, LMNB1 and MATR3 after 6h venetoclax treatment and inhibition of cleavage by Z-VAD-FMK). Thus, the present inventors identified a new method for direct, label free measurement of the cleavage of proteins in cells, while the cells are still intact. Example 2 To further validate the method, the above-described assay (Example 1) was repeated using test samples treated with the following different drugs capable of inducing apoptosis responses: the PI3K inhibitors (PI3Ki) alpelisib and buparlisib; the microtubule-stabilizing taxanes docetaxel and paclitaxel; and methylene quinuclidinone (MQ) the active metabolite of the TP53-stabilizing drug APR-246. The proteins mentioned above, PARP1, MATR3, LMNB1, LMNB2, DDX21 and BCAP31, were analysed. The data is shown in Figure 5. Like venetoclax, AT-IAP resulted in significant RESP effects for all proteins, which demonstrates that the method achieves detection of cleavage of multiple different proteins in the sample, following different cleavage-inducing treatments. This demonstrates that the methods of the invention are not limited to detecting cleavage in a specific protein or proteins, nor to detecting drug-specific cleavage. The method was further shown to detect cleavage of PARP1 following administration of MQ, alpelisib, and bupalisib. After administration of paclitaxel (or docetaxel) and an 8-hour incubation, no protein cleavage was detected. This is consistent with the fact that these drugs do not activate effector caspases after 8 hours of treatment (the durations used in the present study), but only after 20 hours drug treatment (as shown in Figure 6). Example 3 A non-purified test sample was treated with venetoclax for 6 hours, and a non-purified control sample was not drug-treated. Aliquots of the samples were taken, and after heating each aliquot to a different temperature in a series of temperatures (37, 47, 50, 52, 54 and 57°C), soluble and insoluble protein fractions were separated by centrifugation, and the soluble fractions of each heated sample aliquot were prepared for, and analysed by, mass spectrometry. The mass spectrometry data was analysed to determine the level of every peptide in the soluble protein fraction of the heated sample aliquots. For each peptide, at each temperature point, the relative level in the test sample vs. control was determined. At each temperature point, the summed relative levels of each signature peptide in a first domain were calculated, as were the summed relative levels of each signature peptide in a second domain. The summed relative levels were expressed as the summed log2 fold-change of each soluble peptide in the test sample as compared to in the control sample. The results are shown in Figure 1, which shows that the method of the invention successfully determined that four proteins in a non-purified test sample were cleaved. For the four proteins shown, at each heating temperature used, there is a difference between the summed relative levels of signature peptides from a first domain and the summed relative levels of signature peptides from a second domain. For instance, compare the signature peptides at positions 247-322 (first domain) of the protein of interest Q96ST2 with the signature peptides at positions 640-816 (second domain). Likewise, for 060832 (compare peptides at positions 47-424 vs. 434-514), for 075533 (compare peptides at positions 7-252 and 291-1149), and for P42331 (compare peptides at positions 99-498 to 544-632). This indicates that the first domain in each protein displayed a different thermal stability from the second domain as a result of drug-treatment. This indicates that the first and second domains are separated in the test sample, i.e. the four proteins in the test sample were cleaved. Materials and Methods Cell Lines Several cancer cell lines of human origin were purchased from ATCC: MCF7 (HTB-22, derived from pleural effusion at the metastatic site of a 69-year-old female with breast adenocarcinoma), Kasumi-1 (CRL-2724) established from the peripheral blood of an acute myeloid leukemia (AML) patient, and OVCAR-3 (HTB-161) isolated from the malignant ascites of a patient with progressive adenocarcinoma of the ovary. Human acute myeloid leukaemia cell line MOLM16 was from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures. MCF7 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) high glucose medium (D6429, Sigma) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (10500-064, Gibco), Kasumi-1 were maintained in RPMI-1640 medium (R8758, Sigma) supplemented with 20% FBS, OVCAR-3 were maintained in RPMI-1640 medium (R8758, Sigma) supplemented with 20% FBS and M0LM16 were maintained in RPMI-1640 medium (R8758, Sigma) supplemented with 10% FBS. For cell passaging, MCF7 and OVCAR-3 cells were washed with Hanks' Balanced Salt Solution (HBSS) -CaChZ-MgCh (14175-053, Gibco) and detached by 3 minutes incubation with TrypLE Select lx (12563-029, Gibco) at 37°C. Drugs used to induce apoptosis Docetaxel (SI 148, Selleck Chern), Paclitaxel (SI 150, Selleck Chern), Z-VAD-FMK (S7023, Selleck Chern), and methylene quinuclidinone (MQ) (provided by Aprea Therapeutics) were solubilised at 50 mM in DMSO. Venetoclax (S8048, Selleck Chern), Alpelisib (S2814, Selleck Chern). Buparlisib (S2247, Selleck Chern) and AT-IAP (provided by Astex Pharmaceuticals) were solubilised at 10 mM in DMSO. All compound stocks were aliquoted and stored at -80°C. The cellular thermal shift assay (CETSA) Non-purified samples were used. Cells were seeded in the corresponding complete medium at a suitable cell density (previously determined). The cells were then treated with either drug (test sample) or vehicle (control sample) diluted in the corresponding growth medium, and incubated at 37°C and 5% CO2. Adherent cells were briefly washed in HBSS -CaChZ-MgCh, detached from the flask using TrypLE, collected in HBSS +CaC12Z+MgC12, pelleted for 3 min at 300xg, and resuspended in HBSS +CaC12Z+MgC12. Suspension cells were pelleted for 3 min at 300xg, washed twice in HBSS -CaChZ-MgCh, and resuspended in HBSS +CaC12Z+MgC12. Cells corresponding to each treatment condition were aliquoted into PCR tubes then heated in a Veriti thermal cycler (Applied Biosystems) for 3 min at the selected temperatures (between 37-59°C, Table 1). After heating, all samples were subjected to 3 cycles of freeze-thawing in liquid nitrogen in the presence of 1 x Halt™ protease inhibitor cocktail (1861279, Thermo Scientific) or lx Halt™ Protease and Phosphatase Inhibitor Cocktail (1861284, Thermo Scientific). The soluble protein fraction was isolated by 20 min centrifugation at 20 000xg at 4°C, and analysed using either LC-MS or western blotting. For QP samples, 8 M Urea (freshly prepared) with lx Halt™ Protease and Phosphatase Inhibitor Cocktail (1861284, Thermo Scientific) was added to samples, vortexed and incubated for 15 minutes at room temperature and then sonicated. Table 1 Cell line Temperatures Drug Cone. Time point MOLM-16 37C / 47C / 50C / 52C / 54C / 57C AT-IAP 100 nM 4h Kasumi-1 QP / 37C / 47C / 50C / 52C / 54C / 57C Venetoclax 100 nM 6h MCF-7 37C / 47C / 50C / 53C / 55C / 59C Docetaxel 25 pM 8h Paclitaxel QP / 37C / 47C / 50C / 52C / 54C / 57C Alpelisib 10 pM 6h Buparlisib OVCAR-3 37C / 42C / 46C / 52C / 58C APR-246 6 pM 6h Western blot Western blotting was performed on protein extracts obtained by freeze-thawing with 1 x Halt™ Protease and Phosphatase Inhibitor Cocktail (1861284, Thermo Scientific). Protein extract samples were mixed with NuPAGE loading buffer consisting of NuPAGE LDS sample buffer (NP0008, Life technologies) and reducing agent (NP0009, Life Technologies). Proteins were separated on NuPAGE 4-12% Bis-Tris midi gels (WG1403BX10, Invitrogen) for 45-55 min at 200 V. Separated proteins were transferred to nitrocellulose membranes using the iBlot2 system (Invitrogen) and iBlot 2 NC Regular Stacks (IB23001, Invitrogen). Membranes were blocked in 5% (w / v) non-fat milk (Semper AB) in TBS with 0.05% Tween 20 (Medicago 09-7510-100) (TBS-T) for 1 h with gentle shaking. Incubation with primary antibody was performed overnight at 4°C and with gentle shaking. After washing in TBS-T for 3x10 min, the membranes were incubated with secondary antibodies for 1 h, washed again 3x10 min in TBS-T and developed using either Clarity™ Western ECL Substrate (170-5061, BioRad) or Clarity Max™ Western ECL Substrate (1705062, BioRad). All antibodies were prepared in 5% (w / v) non-fat milk in TBS-T at the dilutions listed below. The chemiluminescent signal was detected using the ChemiDoc™ XRS+ imaging system from BioRad and the band intensities were quantified using ImageLab™ software (BioRad). Primary antibodies: 1:1000 anti-cleaved PARP (Asp214) (9541, Cell Signaling), 1:1000 anti-LMNBl N-term (12586, Cell Signaling), anti-MATR3 (abl51714, Abeam), 1:20000 anti-Sodl (HPA001401, Sigma). Sample preparation for LC-MS Protein concentrations were determined in the samples using the DC protein assay (500-0116, BioRad) according to the manufacturer’s protocol and equal amounts of total protein from each condition were used for sample preparation. Samples were dried and resuspended in 50 mM triethylammonium bicarbonate (TEAB) buffer (T7408, Sigma) in H2O (LC-MS grade, 115333, Merck), reduced with 5 mM TCEP (77720, Bond-breaker™, Thermo Scientific) at 65°C for 30 min, followed by alkylation with 15mM 2-chloroacetamide (CAA) (C0267, Sigma) at 37°C in the dark. Digestion of the samples was performed with 1:50 Lys-C (Wako Chemicals Ltd) at 37°C for 2h, followed by 1:50 SOLu-Trypsin (EMS0004, Sigma) at 37°C overnight, for QP samples, before LysC addition amples were diluted to 2M UREA with lOOmM TEAB and before Trypsin addition they were diluted to IM UREA also in lOOmM TEAB. After digestion efficiency was checked, the peptides were labelled with Isobaric Tandem Mass Tags (TMT)-lOplex (90110, Thermo Scientific) or (TMTpro)-16plex (A44522, Thermo Scientific) at 37°C for 3h, followed by checking labelling efficiency (>95% TMT-labelled PSMs). Three biological replicates from the treatment conditions at the same temperature were labelled as a set. The labelled samples from the same TMT set were pooled and the labelling reaction was stopped by adding 10% trifluoroacetic acid (Sigma) to reach pH <3. Samples were dried in a SpeedVac vacuum concentrator (Thermo Scientific) and desalted using Oasis HLB lee (lOmg) extraction cartridges (186000383, Waters) according to manufacturer’s protocol. Offline pre-fractionation of the samples was performed by high pH reverse-phase liquid chromatography using the Xbridge Peptide BEH C18, 300 A, 3.5 pm, 2.1 mm x 250 mm column (#186003610, Waters) The fractions were concatenated and dried using a SpeedVac vacuum concentrator (Thermo Scientific). LC-MS The digested, labeled, and dried peptide sample fractions were resuspended in 0.1% FA (LC-MS grade, 533002, Merck) in H2O (LC-MS grade, 115333 Merck). Online chromatography was performed using Dionex UltiMate 3000 UPLC system coupled to a Q Exactive mass spectrometer (Thermo Scientific). Each fraction was separated on a 50 cm x 75 pm (ID) EASY-Spray analytical column (Thermo Scientific) in a gradient of programmed mixture of solvent A (0.1% formic acid in H2O) and solvent B (99.9% acetonitrile, 0.1% formic acid). MS data were acquired using a top 12 data-dependent acquisition method. Full scan MS spectra were acquired in the range of 375-1500 m / z at a resolution of 60,000 and AGC target of 3e6; Top 12 dd-MS2 60,000 and 3e6 with isolation window at 1.2 m / z. Method for discovery of candidate proteolytically cleaved proteins in MS data sets Peptide and Protein identification and quantification : Protein identification was performed by Proteome Discoverer software (Thermo Scientific), using Sequest HT (Thermo Scientific) search engines to search against reviewed human Uniprot databases (downloaded on 13 Jan 2017, including 42105 sequence entries and another downloaded on 23 Jul 2018, including 9606 sequence entries). MS precursor mass tolerance was set at lOppm, fragment mass tolerance 0.02 Da, and maximum missed cleavage sites of 2. Dynamic modifications searched for Oxidation (M), Deamidation (NQ), and Acetyl N-terminal protein. Static modifications: Carbamidomethyl (C) and TMTIOplex or TMTpro-16plex (K and peptide N terminus). Only the spectrum peaks with signal-to-noise ratio (S / N) >4 were chosen for searches. The false discovery rate (FDR) was set to 1% at both PSM and protein group levels. Only the unique and razor peptides were used for protein assignment and abundance quantification. Isotopic correction of the reporter ions in each TMT channel was performed according to the product sheet. Only the master proteins in the protein group were used for downstream analysis. For each dataset, the peptide abundances were obtained from Proteome Discoverer software (version 2.4). Every peptide with another modification than a TMT one was removed. Algorithm: For all peptides of the dataset, the log2 fold-changes between the venetoclax treated samples and the non-treated samples were obtained. To find potential cleaved proteins from this data, proteins with strictly less than four identified peptides and peptides with strictly more than three missing values are filtered out (50% of the values considering there were six temperatures, hence 6 fold-changes per peptide). These two parameters are arbitrary and can be more stringent. Then, for each protein, the peptide are ordered according their position in the protein amino-acid sequence and each log2 fold-change per peptide is summed so that each peptide has one value. Next, the cumulative sum of the summed log2 fold-changes of every peptide of each protein, respecting the sequence order, is computed. For each protein, this cumulative sum is plotted with relation to the protein amino-acid sequence order and from this curve the R-squared is calculated to evaluate if it has a linear relationship. If it is the case, it can be concluded that every peptide has similar fold-changes, hence that there is no region stabilization effect so that the protein is not cleaved. If not, then two cases can occur, either the peptides located at the beginning of the protein amino-acid sequence have greater fold-changes or the peptides located at the end of the sequence have greater fold-changes. In the first case, the curve obtained from the cumulative sum is concave and in the other case it is convex. In this study, an R-squared cutoff of 0.9 was chosen. To find the potential cleaved site, the inflexion point from the obtained curve of the cumulative sum can be computed to find where the protein is cleaved. If the curve is concave, the minimum of the second derivative of the curve is the inflexion point and if it is convex, then the maximum of the second derivative of the curve is the inflexion point. With this method, cleaved proteins can be detected as well as their cleavage site. To illustrate cleaved proteins, the non-log2 transformed fold-changes of the peptides located before and after the cleaved site were summed separately. Then, these new fold-changes were log2 transformed and plot as bar plots. Quantitative MS data analysis and visualization Quantified protein / peptide abundances were imported into the R environment (http: / / www.R-project.org / ) to facilitate the data analysis and visualization. Data cleaning, normalization, and calculations of protein abundance and thermal stability differences in each condition were performed. Caspase 3 / 7 activity Assay For assessment of caspase 3 / 7 activation after treatment with compounds, MCF7 cells were seeded in complete media in black 96-well polystyrene plates with clear bottom (Corning) at a cell density of 10 000 cells per well. The cells were then incubated overnight at 37° C and 5% CO2 in order to adhere to the plate and resume log-phase growth. The medium was then changed to complete medium containing the fluorescent caspase 3 / 7 reagent (Sartorius) diluted 1:1000 and either vehicle (DMSO) or compound diluted in the complete growth media was added. The cell plate was then incubated and imaged in an IncuCyte S3 live cell analysis system (Essen Bioscience) where phase contrast images and fluorescent images were acquired using the 10x objective lens every 2h for 48h. Number of green objects per image were analyzed using Incucyte 2021A software. Data Analysis and visualisation All graphs were generated using GraphPad Prism and R environment. All data are presented as mean with error bars representing the standard error of the mean (SEM). Error bars that are smaller than the displayed data points are not displayed by the software. Details regarding replicates for each experiment can be found in the figure legends. Sigmoidal curves were fit (where appropriate) using R environment. Unpaired t-tests were performed using GraphPad Prism and the results are displayed in figures and figure legends.

Claims

14 10251. A method for detecting cleavage of a protein of interest in a non-purified test sample, wherein said protein of interest comprises at least a first signature peptide present in a first domain and at least a second signature peptide present in a second domain, wherein the method comprises the steps of:a) heating the non-purified test sample to provide a heated test sample;b) separating soluble from insoluble protein in the heated test sample of step a) to produce soluble and insoluble protein fractions of said heated test sample; andc) determining, in the soluble and / or the insoluble protein fraction of step b), i) a first relative level, which is the level of the first signature peptide as compared to a control level, andii) a second relative level, which is the level of the second signature peptide as compared to a control level,wherein cleavage is detected by a difference between said first and second relative levels in the soluble protein fraction and / or by a difference between said first and second relative levels in the insoluble protein fraction.

2. The method of claim 1, wherein the levels of the first and second signature peptides aredetermined by mass spectrometry or by affinity reagents.

3. The method of claim 1 or claim 2, wherein step b) is a step of centrifugation or filtration.

4. The method of any one of claims 1 to 3, wherein said first and second relative levels aredetermined in the soluble protein fraction of said heated test sample.

5. The method of any one of claims 1 to 4, wherein the method is repeated using one or more different temperatures in step a), and wherein cleavage is detected by a difference between said first and second relative levels after heating to at least one temperature.

6. The method of any one of claims 1 to 5, wherein said method comprises a step of proteolysis after step b) to produce said signature peptides in said protein fraction(s).14 10257. The method of any one of claims 1 to 6, wherein said first and second domains are located either side of a cleavage site in the protein of interest, preferably wherein the cleavage site is a caspase cleavage site.

8. The method of any one of claims 1 to 7, wherein step a) comprisesi) heating the non-purified test sample to a series of temperatures in a temperature range and obtaining an aliquot of the sample after heating at each temperature, orii) obtaining a plurality of non-purified test sample aliquots and heating each sample aliquot to a different temperature within a series of temperatures in a temperature range,to obtain a plurality of heated test sample aliquots;wherein the subsequent steps of the method are performed on each of said heated test sample aliquots,and wherein cleavage is detected by a difference between said first and second relative levels in the soluble and / or insoluble protein fraction at least one of said heated test sample aliquots.

9. The method of claim 8, wherein the method comprisesa) heating the non-purified test sample, wherein said heating step comprisesi) heating the non-purified test sample to a series of temperatures in a temperature range and obtaining an aliquot of the sample after heating at each temperature, orii) obtaining a plurality of non-purified test sample aliquots and heating each sample aliquot to a different temperature within a series of temperatures in a temperature range, to obtain a plurality of heated test sample aliquots;b) separating soluble from insoluble protein in each of the heated test sample aliquots to produce soluble and insoluble protein fractions thereof; andc) determining, in the soluble and / or the insoluble protein fraction of each heated test sample aliquot,i) a first relative level, which is the level of the first signature peptide as compared to a control level, andii) a second relative level, which is the level of the second signature peptide as compared to a control level,wherein cleavage is detected by a difference between said first and second relative levels in the soluble protein fraction of at least one heated test sample aliquot and / or by a difference14 1025between said first and second relative levels in the insoluble protein fraction of at least one heated test sample aliquot.

10. The method of claim 8 or claim 9, wherein step c) further comprises calculating a temperature-summed relative level of the first signature peptide, which is the sum of the first relative levels determined in the soluble protein fractions of each heated test sample aliquot, and calculating a temperature-summed relative level of the second signature peptide, which is the sum of the second relative levels determined in the soluble protein fractions of each heated test sample aliquot, and / orcalculating a temperature-summed relative level of the first signature peptide, which is the sum of the first relative levels determined in the insoluble protein fractions of each heated test sample aliquot, and calculating a temperature-summed relative level of the second signature peptide, which is the sum of the second relative levels determined in the insoluble protein fractions of each heated test sample aliquot,wherein cleavage is detected by a difference between the temperature-summed relative levels of the first and second signature peptides determined in either or both of the soluble and insoluble protein fractions.

11. The method of any one of claims 1 to 10, wherein said protein of interest comprises a plurality of signature peptides in said first domain, and a plurality of signature peptides in said second domain, and wherein step c) comprisesdetermining the relative level of each signature peptide as compared to a control level, wherein cleavage is detected by a difference between the relative level of any one or more signature peptides of said first domain and the relative level of any one or more signature peptides of said second domain, in either or both of the soluble and insoluble protein fractions of said heated test sample or of at least one heated test sample aliquot.

12. The method of claim 11, wherein the method comprisesa) heating the non-purified test sample, wherein said heating step comprisesi) heating the non-purified test sample to a series of temperatures in a temperature range and obtaining an aliquot of the sample after heating at each temperature, orii) obtaining a plurality of non-purified test sample aliquots and heating each sample aliquot to a different temperature within a series of temperatures in a temperature range,to obtain a plurality of heated test sample aliquots;14 1025b) separating soluble from insoluble protein in each of the heated test sample aliquots to produce soluble and insoluble protein fractions thereof; andc) determining the relative level of each signature peptide in the soluble protein fraction of each heated test sample aliquot, as compared to a control level of said signature peptide, and / ordetermining the relative level of each signature peptide in the insoluble protein fraction of each heated test sample aliquot, as compared to a control level of said signature peptide;wherein cleavage is detected by a difference between the relative level of any one or more signature peptides of said first domain and the relative level of any one or more signature peptides of said second domain, in either the soluble and / or in the insoluble protein fraction of at least one heated sample aliquot.

13. The method of claim 11 or claim 12, wherein the determined relative levels of each signature peptide of the first domain are summed to produce a first summed relative level, and the determined relative levels of each signature peptide of the second domain are summed to produce a second summed relative level,wherein cleavage is detected by a difference between the first and second summed relative levels in either or both of the soluble and insoluble protein fractions of said heated test sample, or of at least one heated test sample aliquot.

14. The method of claim 12, wherein step c) further comprises,i) calculating a temperature-summed relative level for each signature peptide, which is the sum of the relative levels of the signature peptide determined in the soluble protein fraction of each heated test sample aliquot,wherein cleavage is detected by a difference between the calculated temperature-summed relative level of one or more signature peptides of said first domain and the calculated temperature-summed relative level of one or more signature peptides of said second domain;and / orii) calculating a temperature-summed relative level for each signature peptide, which is the sum of the relative levels of the signature peptide determined in the insoluble protein fraction of each heated test sample aliquot,wherein cleavage is detected by a difference between the calculated temperature-summed relative level of one or more signature peptides of said first domain and the calculated temperature-summed relative level of one or more signature peptides of said second domain.14 102515. The method of claim 11, 12 or 14, wherein the method further comprises locating acleavage site in the protein of interest by:a) ordering the determined relative levels or temperature-summed relative levels of the signature peptides on the basis of the sequence alignment of the signature peptides with the protein of interest, in either the N-terminus to C-terminus direction or the C-terminus to N-terminus direction,wherein a cleavage site is located in the protein of interest between the two adjacent signature peptides between which there is the largest difference in the determined relative levels or temperature-summed relative levels; orb) ordering the cumulative differences in the temperature-summed relative levels of the signature peptides on the basis of the sequence alignment of the signature peptides with the protein of interest, in either the N-terminus to C-terminus direction or the C-terminus to N-terminus direction,wherein a cleavage site is located in the protein of interest by the presence of an inflexion point between two adjacent signature peptides.

16. A method for detecting cleavage of a protein of interest in a non-purified test sample, wherein said protein of interest comprises at least a first signature peptide present in a first domain and a second signature peptide present in a second domain,wherein the method comprises the steps of:a) heating the non-purified test sample to provide a heated test sample; orb) heating the non-purified test sample to provide a heated test sample and separating soluble from insoluble protein in the heated test sample to produce soluble and insoluble protein fractions of said heated test sample;and subsequentlyc) determining, in said heated test sample, or in the soluble and / or insoluble protein fraction thereof,i) a first relative level, which is the level of the first domain as compared to a control level, andii) a second relative level, which is the level of the second domain as compared to a control level,wherein cleavage is detected by a difference between said first and second relative levels in said heated test sample, or in said soluble and / or insoluble protein fraction thereof.14 102517. The method of claim 16, wherein the method comprisesa) heating the non-purified test sample, wherein said heating step comprisesi) heating the non-purified test sample to a series of temperatures in a temperature range and obtaining an aliquot of the sample after heating at each temperature, orii) obtaining a plurality of non-purified test sample aliquots and heating each sample aliquot to a different temperature within a series of temperatures in a temperature range,to obtain a plurality of heated test sample aliquots; orb) heating the non-purified test sample, wherein said heating step comprisesi) heating the non-purified test sample to a series of temperatures in a temperature range and obtaining an aliquot of the sample after heating at each temperature, orii) obtaining a plurality of non-purified test sample aliquots and heating each samplealiquot to a different temperature within a series of temperatures in a temperature range, to obtain a plurality of heated test sample aliquots and separatingsoluble from insoluble protein in each of the heated test sample aliquots to produce soluble and insoluble protein fractions thereof;and subsequentlyc) determining, in each heated test sample aliquot, or in the soluble and / or insoluble protein fraction thereof,i) a first relative level, which is the level of the first domain as compared to a control level, andii) a second relative level, which is the level of the second domain as compared to a control level,wherein cleavage is detected by a difference between said first and second relative levels in the heated test sample, or soluble and / or insoluble protein fraction thereof, or in at least one heated test sample aliquot, or soluble and / or insoluble protein fraction thereof.

18. The method of claim 16 or claim 17, wherein step c) further comprisescalculating a temperature-summed relative level of the first domain, which is the sum of the first relative levels determined in each heated test sample aliquot, or in each soluble protein fraction thereof, or in each insoluble protein fraction thereof, andcalculating a temperature-summed relative level of the second domain, which is the sum of the second relative levels determined in each heated test sample aliquot or in each soluble protein fraction thereof,14 1025wherein cleavage is detected by a difference between the temperature-summed relative levels of the first and second domains.

19. The method of any one of claims 16 to 18, wherein the levels of the first and second domains are determined by affinity reagents.

20. The method of any one of claims 16 to 19, wherein the levels of the first and second domains are determined in said heated test sample or heated test sample aliquot, or the soluble protein fraction thereof, by affinity binding using at least two affinity reagents specific for the soluble form of the domain.

21. The method any one of claims 1 to 20, wherein said heating step a) comprises heating to a series of temperatures in the temperature range of 37°C to 60°C.

22. The method of any one of claims 1 to 21, wherein said test sample has been exposed to a test condition, and wherein said control level is the level of said signature peptide or domain in an equivalent heated non-purified control sample or sample aliquot, or protein fraction thereof, wherein said control sample has not been exposed to said test condition.

23. The method of any one of claims 1 to 22, wherein each of said differences is a statistically significant difference.

24. The method of any one of claims 1 to 24, wherein the protein of interest is selected from the group consisting of PARP1, DDX21, LMNB2, LMNB1, BCAP31 and MATR3.

25. The method of any one of claims 1 to 24, wherein the protein of interest is within or on a cell.

26. A method of detecting apoptosis in a cell in a non-purified test sample, wherein the method comprises detecting cleavage of a protein of interest within or on the cell, by performing the method of any one of claims 1 to 25,wherein the protein of interest is selected from the group consisting of PARP1, DDX21, LMNB2, LMNB1, BCAP31 and MATR3;and wherein apoptosis is detected if cleavage of one or more of said proteins of interest is detected.14 1025

Images