Ms-detectable peptide tags

Abstract

This invention provides methods and use of uniquely identifiable peptide tags for protein detection using mass-spectrometry detection methods.

Description

[0001] TITLE: MS-DETECTABLE PEPTIDE TAGS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the detection and quantification of proteins in a sample, relying on standard mass spectrometry techniques. Specifically, it provides uniquely identifiable peptide tags with an inherent signal-amplification feature, methods for sample preparation, their application in proteomics, and a kit of parts.

[0004] BACKGROUND OF THE INVENTION

[0005] Mass Spectrometry (MS)-based proteomics has become a mature experimental tool for studying protein dynamics in various sample types, including cell lines, primary samples, and biopsy samples such as plasma, tissue, urine, and spinal fluid. Recent advancements have led to the development of single-cell proteomics by Mass Spectrometry (scp-MS). This technology enables the detection and quantification of thousands of proteins in individual cells. Currently, proteomics using standard MS methods can be either untargeted or targeted.

[0006] Untargeted, or global proteomics, is the data driven hypothesis-free analysis of (nearly) all proteins expressed and quantitation thereof in a sample, but with no guarantee of measuring specific proteins of interest, and with greater difficulty of capturing rare or low expressed proteins. CyTOF is the current state-of-the-art method for detecting rare proteins in low-abundance samples, it involves the use of antibodies labelled with lanthanide metals. However, this approach is limited by the number of available lanthanides metals and requires specialized, expensive equipment, restricting its widespread use.

[0007] Targeted proteomics is the hypothesis-driven analysis of a subset of proteins, typically involving preselected targets in the range of lOs-lOOs of proteins and quantitation thereof, rather than unbiased global analysis. Targeted proteomics provides high sensitivity and quantitative accuracy, but only of the proteins that are actively sought. Targeted proteomics relying on antibody-based technologies, among others, utilize mass tags coupled to antibodies, as described in US 2012 / 0196297 Al. The technology, that which has been known for more than a decade, uses antibodies linked to a mass tag and then using the mass tag signature to detect the antibody in the sample. Mass tags can be chemical moieties with a mass of 100-2000Da, and may comprise structures that include carbon, nitrogen, oxygen, hydrogen, fluorine, sulphur, phosphorus, and iodine. Other types of mass tags such as a peptide, a nucleic acid, a gold particle, a sugar or a fluorochrome are presented in US 8,221,972 B2. WO 2024 / 141759 Al describe a mass tag for recovery and identification of a target protein using MS, wherein the tags can be multiplexed to allow for samples to be pooled. The mass tags of WO 2024 / 141759 Al provide diagnostic fragment ions due to selective fragmentation. WO 2024 / 108178 Al present an immunoaffinity capture assay, in which an analyte of interest is first captured and subsequently detected using a detection reagent (e.g. an antibody) comprising a cleavable linker bearing multiple MS-detectable tags, which are released and quantified by LC-MS / MS. These mass tags and those otherwise described enable detection of specific proteins that have been isolated or affinity-captured from a sample using MS-based readout approaches, rather than constituting a bottom-up proteomics workflow. In contrast, bottom-up proteomics refers to enzymatic digestion of proteins into peptides followed by LC-MS / MS analysis. Currently standard bottom-up proteomics does not readily enable highly sensitive, affinity-guided detection and quantification of specific low-abundance proteins while simultaneously preserving broad proteome coverage within a single integrated measurement.

[0008] SUMMARY OF THE INVENTION

[0009] In accordance with the purpose of this invention, the present application discloses uniquely identifiable peptide tags, their use and a method relating thereto, as well as a kit of parts.

[0010] The invention addresses the objective technical problem of sensitive, specific, and quantitative detection of selected proteins, including low-abundance proteins, within complex biological samples sample using mass spectrometry (MS) techniques. The invention additionally solves the problem of precisely detecting and quantifying specific proteins, including rare or low-abundance proteins, in a complex sample using mass spectrometry (MS) techniques.

[0011] The invention circumvents limitations related to the current state of the art by using uniquely identifiable peptide tags attached to a molecule with specific binding affinity (a molecule comprising at least one binding moiety) for a protein of interest in the sample and thus enables a highly sensitive and indirect detection of the protein of interest using MS techniques. The use of peptide tags further integrates into a standard bottom-up proteomic approach. Specifically, the peptide tags are designed to be released during proteolytic digestion (e.g. trypsin digestion), thereby generating reporter peptides alongside endogenous sample-derived peptides. Thus, this approach enables signal amplification proportional to the number of peptide repeats while allowing simultaneous analysis of both the proteins of interest and the broader proteome within the same LC-MS / MS workflow.

[0012] One aspect of the invention is the use of a molecule with specific binding affinity (a molecule comprising at least one binding moiety) for a protein or other biological molecule of interest that is linked and / or fused and / or conjugated with a peptide tag, wherein the peptide tag comprises two or more serially linked repetitive peptide sequences that are releasable upon proteolytic digestion. Or releasable upon another method such as fragmentation, proteolytical cleavage and non-enzymatic digestion. The peptide sequence has a sequence which is uniquely identifiable by standard MS-detection methods. The MS-detected signal of the peptide tag may be directly proportional to the absolute number of repetitive peptide sequences in the peptide tag. Thus, allowing the quantification of the total number of proteins of interest detected by the molecule with specific binding affinity (a molecule comprising at least one binding moiety) for the protein of interest detected in the sample. The MS-detected signal of the peptide tag correlates with the number of repetitive peptide sequences present in the tag and the abundance of the bound target molecule. Thus, enabling quantitative determination of the amount of the protein of interest in the sample via the detected reporter peptide signal.

[0013] The design of the repetitive peptide sequence comprised in the peptide tag may be a peptide sequence non-native to the sample. Optionally it may be a peptide sequence native to the sample.

[0014] The sequence length of the repetitive peptide sequence comprised in the peptide tag may be selected from a range such as 1-5 amino acids, 5-10 amino acids, 10-15 amino acids, 15-20 amino acids, 20-25 amino acids, 25-30 amino acids, 30-35 amino acids, preferably 6-25 amino acids.The serially linked repetitive peptide sequences have the technical effect of amplifying the detected signal.

[0015] The serially linked peptide sequences comprising the peptide tag may be reversible attached to each other. The reversible attachment between the repetitive peptide sequences may be sensitive to proteolytic cleavage. Optionally the link between the repetitive peptide sequences may comprise one or more amino acids, that is not forming part of the repetitive peptide sequence.

[0016] The number of serially linked repetitive peptide sequence comprised in the peptide tag sequences may be between 2 and 120, such as 2 copies, such as 3 copies, such as 4 copies, such as 5 copies, such as 6 copies, such as 7 copies, such as 8 copies, such as 9 copies, such as 10 copies, such as 11 copies, such as 12 copies, such as 13 copies, such as 14 copies, such as 15 copies, such as 16 copies, such as 17 copies, such as 18 copies, such as 19 copies, such as 20 copies, such as 21 copies, such as 22 copies, such as 23 copies, such as 24 copies, such as 25 copies, such as 26 copies, such as 27 copies, such as 28 copies, such as 29 copies, such as 30 copies, such as 31 copies, such as 32 copies, such as 33 copies, such as 34 copies, such as 35 copies, such as 36 copies, such as 37 copies, such as 38 copies, such as 39 copies, such as 40 copies, such as 41 copies, such as 42 copies, such as 43 copies, such as 44 copies, such as 45 copies, such as 46 copies, such as 47 copies, such as 48 copies, such as 49 copies, such as 50 copies, such as 55 copies, such as 60 copies, such as 65 copies, such as 70 copies, such as 75 copies, such as 80 copies, such as 85 copies, such as 90 copies, such as 95 copies, such as 100 copies, such as 105 copies, such as 110 copies, such as 115 copies, such as 120 copies or a variation thereof.

[0017] A first end of a first peptide tag may be reversibly attached by linking and / or fusing and / or conjugation to one or more sites on the molecule with specific binding affinity for a protein of interest.

[0018] A first end of a peptide tag may be attached by being linked and / or fused and / or conjugated to one or more sites on the molecule with specific binding affinity (a molecule comprising at least one binding moiety) for a protein or biological molecule of interest.

[0019] If the molecule with specific binding affinity (a molecule comprising at least one binding moiety) for a protein of interest may be or is an antibody or antibody binding member a first end of a first peptide tag may be linked and / or fused and / or conjugated to one or more heavy and / or light chains.

[0020] Optionally, to each heavy chain and / or light chain of the antibody or antibody binding member. Optionally, at the N-terminal and / or C-terminal end of the antibody or antibody binding member. Optionally, at the N-terminal and / or C-terminal end of the heavy chain of the antibody or antibody binding member. Optionally, at the N-terminal end of the heavy chain of the antibody or antibody binding member. Optionally, at the C-terminal end of the heavy chain of the antibody or antibody binding member.

[0021] The linking and / or fusing and / or conjugation to the molecule with specific binding affinity for a protein of interest and / or the link between the peptide tag may be amenable to enzymatic cleavage during enzymatic digestion treatment or released by other means. Optionally the peptide cleavage treatment is denaturation, cleavage by a protease and / or a chemical agent, or any other means of peptide cleavage.The sample may be a single cell, derived from mammals, plants, eukaryotes and prokaryotes.

[0022] Optionally the sample may comprise a plurality of cells, derived from mammals, plants, eukaryotes and prokaryotes. Optionally the sample is biofluids, cell lysates, tissue biopsies, preserved clinical specimens, or fresh material derived from mammals, plants, eukaryotes and prokaryotes.

[0023] Optionally an additional fluorophore, and / or chromogen, and / or metal particle and / or other imaging-based detection agent may be conjugated or attached to the molecule comprising the binding moiety (molecule with specific binding affinity) beyond the signal amplification peptide tag. In another aspect the invention describes a method for detecting the number of a protein- and / or molecule-of-interest in a sample, the method comprising;

[0024] a) providing a biological sample that comprises one or more proteins of interest,

[0025] b) contacting the sample of a) with a molecule comprising at least one binding moiety linked and / or fused and / or conjugated to a peptide-tag as defined in the invention.

[0026] c) adding a proteolytic agent such as an enzyme e.g. trypsin to the mixture of a) and b), whereby the conjugated peptides (serially linked repetitive peptide sequences) are digested, optionally applying another method of releasing the peptides sequences from the peptide tag such as fragmentation or non-enzymatic cleavage,

[0027] d) detecting the digested peptides by any suitable technique based on mass-spectrometry, wherein the signal obtained from the peptide sequence correlates to the quantity of protein-of-interest in the sample a).

[0028] In another aspect the invention describes a kit of parts wherein the first end of the peptide tag comprises a reactive group configured to link to the molecule comprising at least one binding moiety, and wherein a coupling reagent suitable for facilitating the linkage between the peptide tag and the molecule comprising at least one binding moiety is provided. Such solution has the benefit of providing ready solutions to link a peptide tag to any suitable molecule comprising at least one binding moiety. In another aspect the invention describes an analyte detection system comprising a) fusion protein comprising a peptide tag domain, and a moiety binding domain; and b) a nuclear acid molecule encoding the fusion protein of a), and wherein the system may be expressed in an eukaryotic, prokaryotic, or cell-free expression system.

[0029] BRIEF DESCRIPTION OF FIGURES

[0030] Figure 1 Illustrates an overview of the peptide tag design and experimental section. pcDNA3.1 vectors including the insertion site for the peptide of interest. Schematic representation of the produced peptide tags targeting either PDL1 or HER2, with peptide tags with increasing numbers of repetitive peptide sequences. Peptide tag labeling used for in vitro characterization assays and MS. Representation of the double positive single cells sorted using two different peptide tags, digestion and MS analysis for peptide identification.Figure 2 Illustrates the in vitro characterization of the peptide tags. (A) In vitro binding characterization for PDL1 antibody linked with a peptide tag comprising 2, 16 and 32 repetitive peptide sequences. Peptide tagged aPDLl were tested in increasing concentrations with PDL1+ and PDL1- cell lines. Human aPDLl IgG is shown as positive control for the binding. (B) In vitro binding characterization for. HER2 antibody linked with a peptide tag comprising 16 and 32 repetitive peptide sequences. HER2 peptide tags were tested in increasing concentrations with HER2+ and HER2- cell lines. Human aHER2 IgG is shown as positive control for the binding.

[0031] Figure 3 Illustrates the increased sensitivity through peptide tags comprising repetitive peptide sequences conjugated to the PDL1 antibody. Calibration curve of the peptide tagged PDL1 antibody conjugated to peptide tags comprising 2 and 32 repetitive peptide sequences respectively. Equal amounts of peptide tagged PDL1 antibody were digested with trypsin and the peptide tags were acquired by targeted MS in a stable HeLa digest matrix. A higher overall signal of the 32-tag repeats was observed at the same antibody concentration and a lower limit of quantification (LOQ.) was determined.

[0032] Figure 4 Illustrates a peptide tagged PDL1 and HER2 antibodies bind to single cells and are detectable with MS. (A) Single cells stained with PDL1 antibody conjugated with peptide tags comprising 0, 2 and 32 repetitive and serially linked repeats. Peptide tag expression detected on single cells. ( B) Single cells stained with HER2 antibodies conjugated with peptide tags comprising either 0 or 16 repetitive peptide sequences . Peptide tag expression detected on single cells. (C) Correlation between expression of the PLD1 antibody on single cells detected by fluorescence (FACS) and targeted-MS. (D) Correlation between expression of HER2 antibodies on single cells detected by fluorescence (FACS) and targeted-MS. (E) Numberof antibodies bound to each cell based on the number of repetitive peptide sequences. (F) Correlation between the expression of PDL1 antibodies on single cells detected by fluorescence (FACS) and label free DIA-MS. (G) Number of proteins identified on each single cell beyond the peptide tags. Mean and standard error displayed. Cells with identified tag peptides for HER2 or PDL1 highlighted by shape

[0033] Figure 5 Illustrates cells double-stained with both a peptide tag of 32 repetitive peptide sequences tagged PDL1 antibodies and peptide tag of 16 repetitive peptide sequences tagged HER2 antibodies. (A) PDL1 expression on 1, 2 and 4 cells, which displayed expression of both PDL1 and HER2 based on the antibodies, as detected by standard FACS correlated to targeted-MS quantification of the peptide tags associated with the PLD1 antibody. (B) HER2 expression on 1, 2 and 4 cells which displayed expression of both PDL1 and HER2 based on the antibodies as detected by standard FACS correlated to targeted-MS quantification of the peptide tags associated with the HER2 antibody. (C) Number of proteins identified on 1, 2 and 4 cells beyond the tag-peptides. Mean and standard error displayed.

[0034] DETAILED DESCRIPTION OF THE INVENTION

[0035] The invention addresses the objective technical problem of precisely detecting and quantifying a number of specific proteins including rare or low abundance proteins as well as other biological molecules for which a molecule with a specific binding affinity exist in a sample using mass spectrometry (MS) techniques. The invention circumvents limitations related to the current state of the art by using uniquely identifiable peptide tags attached to a molecule with specific binding affinityfor a protein of interest in the sample and thus enables a highly sensitive and indirect detection of e.g., the protein of interest using MS techniques. The use of peptide tags further integrates into a standard bottom-up proteomic approach. Thus, providing amplification of the signal relating to the proteins of interest including total quantification of the number of these while allowing for the determination of the protein make-up in a sample, within the same workflow.

[0036] A peptide tag comprises one, two or more serially linked repetitive peptide sequences reversible attached to each other. The peptide sequence comprises a known sequence of amino acid polymers that may, or may not, be found in the sample. Each peptide sequence can be serially linked in repetitive repeats whereby a peptide tag can comprise one, two or more of the same uniquely identifiable peptide sequence linked in series. The peptide sequences can be serially linked by for example continuous repetition of the unique identifiable peptide tag sequence or with a link comprising one or more animo acids in between each repetition of the sequence.

[0037] A potentially unlimited number of peptide sequences can be designed for example by selecting known peptide sequences from phylogenetic different species than the one where the sample at hand originates or through synthetic design. The design of uniquely identifiable peptide sequences will be within the capability of the person skilled in the art, once presented with the information disclosed in this document and exemplified in the examples.

[0038] The first end of a peptide tag , may be attached or anchored to a molecule with at least one specific binding affinity to a protein of interest, such a molecule being in non-limiting examples, an antibody or a single domain antibody or an antibody binding member, or a mini-bodies or a DARPin or an armadillo repeat protein or a bicyclic peptides, such as including but not limited to full length antibody, scFv-Fc, sdAb-Fc, nanobody-Fc, Fab, scFv, sdAb, nanobody, or another antibody fragment, minibinder, triabody, tetrabody, dAbs-Fc, Fab, scFv, (scFv)2, dAbs, VHH, diabody, sdAb, nanobody, scDb and taF.The means for attaching or anchoring a peptide sequence to another peptide sequence through, for example fusing or conjugation is well established within the state of the art and is within the capability of the person skilled in the art. Attaching or anchoring the peptide tag to such a molecule, enables the peptide tag to be associated with the protein or biological molecule of interest once subjected to a sample containing the protein of interest.

[0039] The attachment or anchoring of the first end of a peptide tag to the molecule with at least one specific binding affinity to a protein of interest can be through genetic fusion as a fusion protein or through chemical coupling using known technologies such as click chemistry or biotinylation.

[0040] The serially linked repetitive peptide sequences comprised in the peptide tag can be linked directly or via linkers, with genetic fusion being preferable. There can be an additional linker or amino acid(s) between the binding molecule and the peptide-tag / repetitive peptide tags. This may be necessary for the conjugation technique or advantageous for spacing etc for the fusion technologies. This may be the same at each position of fusion, but it may also be different, e.g. a certain longer flexible linker to the N-term of the heavy chain and no or a short for the C-term. This may need to be compared and optimized during development in order to achieved suitable binding and the sensitivity, and is within the standard optimization work of a person skilled in the art.

[0041] The attachment between the peptide tag and the molecule comprising a specific binding affinity may be designed to be sensitive to fragmentation such as by enzymatic cleavage, chemical cleavage,physical fragmentation, electrochemical and catalytic methods or a combination. Enzymatic cleavage includes the current state of the art in proteolytic cleavage, such as trypsin or Lysyl Endopeptidase C. Physical fragmentation may include collision-induced-, PSD-MALDI-, higher energy C-trap-, electron transfer-, electron capture-, in source-, post-source and ultraviolet photo-dissociation. Likewise, the link between the repetitive peptide tag sequences can be designed to be sensitive to fragmentation in the same or different manner as the attachment between the peptide tag and the molecule. The type of fragmentation to which the repetitive peptide tags and the peptide tag to the molecule comprising a specific binding affinity are sensitive can be optimized for the method and purpose and is within the skills of the person skilled in the art once presented with the information and examples.

[0042] A technical effect of a peptide tag comprising serially linked repetitive peptide sequences anchored to a molecule comprising a specific binding affinity domain for a protein of interest is the indirect signal amplification of the protein of interest. For example, by linking a peptide tag comprising 16 repetitive peptides sequences to the N-terminal end to each of the heavy chains of a standard antibody would, after the antibody has bound to the protein of interest and the sample subjected to enzymatic digestion or another alternative methods of releasing the peptide tag and repetitive peptide sequences comprised in the peptide tag, each antibody binding event would result in a 32-fold increase in signal in standard MS analysis compared to having a peptide tag comprising one peptide sequence linked or even just the protein itself.

[0043] An additional technical effect of a peptide tag comprising serially linked repetitive peptide sequences anchored to a molecule comprising a specific binding affinity domain for a protein of interest is the ability to indirectly quantify the number of proteins of interest in the sample. For example, as presented above, a standard antibody with a peptide tag comprising 16 serially linked repetitive peptide sequences to each of its heavy chains would result in a 32-fold increase of the signal compared to a tag comprising a single peptide sequence. Given that each antibody will bind to one protein of interest, knowing the signal size of each peptide sequence in a peptide tag and how many peptide tags are attached to each antibody will enable the indirect calculation of the total number of protein of interests detected in a sample. The example extends beyond antibodies, to other molecules comprising a specific binding affinity domain for a protein or other biological molecule of interest. An essential part of the invention is the ability to design and develop a large number of peptide tags that seamlessly and unambiguously can be detected along with the standard analysis of proteins in a sample using established and readily available analytical methods. Thus, providing a solution where bottom-up proteomics can be combined with total quantification of selected proteins of interest using the peptide tags reversibly linked to a molecule with a biding affinity to the protein of interest. Different peptide tags each comprising a different repetitive peptide sequence reversible linked to different molecules with binding moieties specific for different proteins of interest enable a true multiplexing potential achieving total quant for a plurality of targets in a sample. The approach can also be used to identify proteins of interest in samples, that after contact with a peptide tag, are pooled. This may be achieved by attaching different peptide tags each comprising a different repetitive peptide sequence reversible linked to molecules with binding moieties specific to the same protein of interest and contacting the peptide-tagged molecule to the sample prior to pooling samples.

[0044] Several options to attain a, for the sample unique, sequence exist. Options for identifying sequences unique to a sample include selecting from a different phylogenetic branch, different species or viasynthetic design of peptides. Alternatively, the repetitive peptide sequence may be selected from known control peptides. Such control peptide may serially linked into a peptide tag of varying length, allowing for amplification and a well-known m / z signature across MS techniques and even machines. The optimal sequence length for a repetitive peptide sequence in a peptide tag varies, inventors have found that a length between 6 and 25 amino acids work well, but shorter and longer tags are possible, with shorter being limited mainly by difficulties in uniqueness and longer limited by the current mass spectrometric standards.

[0045] A suitable peptide tag can be detected from genomic, transcriptomic or proteomic analysis of the target cell or species of interest and compared to those which are unequal and exclusive to the target cell or species of interest. This delivers potentially available protein sequences directly or deductible from genetic information. From the protein sequences sufficiently different peptide sequences, which able to differentiate between the target cell or species of interest from those of the source of the peptide, can be deducted, ideally those which can be generated through enzymatic digestion and hence certain amino acid position.

[0046] The suitability of a repetitive peptide sequence to work as a peptide-tag (or repetitive peptide-tag) as defined in the invention, can be identified by testing their ability to fly (ability to ionize) to garment and to be identified by MS as well as their ability to deliver a potentially desired amplification of and quantification of the signal.

[0047] Global proteomes from the animal kingdom can be used as a source for identifying and selecting repetitive peptide sequences or peptide tag sequences, as previous MS-detectability is a good indicator of ionization. Besides these naturally derived peptides there can also be those synthetically generated which are different from the ones present in the target cell or species of interest. These synthetically generated peptide-tags can be either achieved by full synthetic composure or by adding, deleting or mutating amino acids in a naturally occurring peptide. The peptide tag can also be heavy labeled (through incorporation of heavy isotopes) to sufficiently differentiate from naturally occurring counterparts.

[0048] Alternatively, repetitive peptide sequence suitable for use in a peptide tag may be selected to have a mass-to-charge ratio (m / z) and elution time (as determined by peptide hydrophobicity) that is distinct from the m / z values and elution time of endogenous peptides present in the sample. Further, the repetitive peptide sequence is selected such that, upon fragmentation, the resulting monomers occupy a predetermined and non-overlapping m / z window in a mass spectrum.

[0049] The benefits of this invention span applications from e.g. but not limited to bulk proteomics, cellular sub-population analysis, regions-of-interest from solid tissues (e.g., by means of laser capture microdissection), body- or other fluid analysis, and single-cell proteomics by mass spectrometry (scp-MS). The peptide tag technology allows staining of live cells and tissue samples but can even stain specific proteins in liquid biopsies or cellular lysates by means of immunoprecipitation or proteinaggregation capture (PAC) methods. Optionally the molecule comprising a specific binding affinity for a protein of interest can, beyond the peptide tag, be conjugated to a fluorophore, chromogen or other imaging-based detection agent, to combine imaging-based analysis (e.g. immunofluorescence microscopy or fluorescence-activated cell sorting, FACS) with MS-based detection and quantification of tag peptides.In another aspect, the invention relates to a peptide tag wherein the first end of the peptide tag comprises a reactive group configured to link to the molecule comprising at least one binding moiety. The reactive group is compatible with click chemistry, such as an azide or alkyne. A peptide tag wherein the first end comprises a reactive group may be in a kit wherein a coupling reagent suitable for facilitating the linkage between the peptide tag and the molecule comprising at least one binding moiety is provided. Such solution has the benefit of providing ready solutions to link a peptide tag to any suitable molecule comprising at least one binding moiety.

[0050] In another aspect, the invention describes an analyte detection system comprising a) fusion protein comprising at least one peptide tag domain, and a binding moiety domain; and b) a nuclear acid molecule encoding the fusion protein of a), and wherein the system may be recombinantly expressed in a eukaryotic, prokaryotic, or cell-free expression system. Wherein the at least one peptide tag domain comprises repetitive peptide sequences, and wherein the repetitive peptide sequences may be serially linked. Further, wherein the length of the peptide sequences in the at least one peptide tag domain may be between 1 and 35 amino acids such as 1-5 amino acids, 5-10 amino acids, 10-15 amino acids, 15-20 amino acids, 20-25 amino acids, 25-30 amino acids, 30-35 amino acids, preferably 6-25 amino acids; and wherein the number of repeated peptide sequences are an integer between 2 and 120. Further, wherein the binding moiety domain is an antibody, single domain antibody or any other molecule with binding affinity to a protein of interest.

[0051] In a further aspect, the peptide tag according to the invention, may be a repetitive peptide sequences configured to be cleaved by a protease during sample preparation, preferably by trypsin digestion, to release one or more mass spectrometry-detectable peptide fragments.

[0052] In another aspect the peptide tag according to the invention may comprises at least two repetitive peptide sequences, preferably between 2 and 20, more preferably between 4 and 12, such that the detected MS signal is proportional to the number of repetitive sequences.

[0053] In yet a further aspect, the peptide tag according to the invention may be a repetitive peptide sequences which may be selected to produce detectable precursor and / or fragment ions under liquid chromatography-mass spectrometry (LC-MS / MS) conditions, and are of a length between 4 and 25 amino acids, preferably between 6 and 20 amino acids.

[0054] Definitions

[0055] Throughout the present specification and the accompanying claims, the word "comprise" and variations such as "comprises" and "comprising" are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows, and sets out a minimum condition without excluding additional elements. The term "molecule comprising at least one binding moiety" or "a molecule comprising a specific binding affinity domain for a protein of interest", "a molecule comprising a specific binding affinity domain for a protein or biological molecule of interest" or " a molecule comprising a specific binding affinity for a protein of interest" as used herein, refers to molecules with specific binding affinity towards a biological molecule, preferentially proteins or peptides, glycoproteins or glycopeptides or other postranslationally modified proteins (phosphorylation, ubiquitinylation etc.), but also againstcarbohydrates, lipids or glycolipids, glycosaminoglycans etc. and include in non-limiting examples, antibodies, single domain antibodies, antibody binding member, mini-bodies DARPin, armadillo repeat protein or bicyclic peptides, scFv-Fc, sdAb-Fc, nanobody-Fc, Fab, scFv, sdAb, nanobody, or another antibody fragment, minibody, triabody, tetrabody, dAbs-Fc, Fab, scFv, (scFv)2, dAbs, VHH, diabody, sdAb, nanobody, scDb and taF.

[0056] An analyte is a biological molecule, preferentially proteins or peptides, glycoproteins or glycopeptides or other posttranslational modified proteins (phosphorylation, ubiquitinylation etc.), but also against carbohydrates, lipids or glycolipids, glycosaminoglycans etc.

[0057] The term "peptide" as used herein, refers to an amino acid polymer wherein each amino acid is linked to its neighbor by an amide bond -C(=O)NH-, also called peptide bond.

[0058] The term "peptide tag" or "AbTag" as used herein, refers to a peptide comprising one, two or more serially linked repetitive peptide sequences, wherein the repetitive peptide sequences is a known and uniquely identifiable sequence of amino acids that can be uniquely identified in the sample of interest. The signal obtained from the peptide tag is directly proportional to the number of serially linked repetitive peptides sequences in a tag. The repetitive peptide sequence or amino acid polymer sequences can be serially linked in repetitive repeats making up peptide tags of variable length. Each repetitive peptide sequence comprised in a peptide tag may be considered a peptide tag per se, which when serially linked become a peptide tag comprising a plurality of peptide tags. In another aspect, the definition comprises a peptide which is detectable as a distinct precursor ion, and / or produce identifiable fragment ions, and / or is separable from background proteome.

[0059] The terms "peptide copy number", "serially-linked peptide-tag", "repetitive peptide sequence", "tandem-repeats", "peptide tag repeats" and "repetitive and serially linked repeats" as used herein, are used to describe the peptide tags of variable length that is the repetitive peptide sequence comprised in the tag also known as an amino acid polymer sequences when they are configured as serially linked in repetitive repeats. However, the use of "peptide tags" and "repetitive and serially linked repeats "should be interpreted inclusively, e.g., the first end of a peptide tag includes both the first end peptide tag consisting of one peptide tag as well as a peptide tag comprising two or more repetitive tags serially-linked.

[0060] The term "conjugated", "linked", "fused", "attached" and "anchored" as used herein is to be interpreted in it most inclusive manner and refers to all possible genetic, chemical, enzymatic and molecular means by which a person skilled in the art can connect two peptide polymer sequences to one another. The connection may be optimized for the sample and detection need, the connection can be the same or different between the repetitive peptide sequences comprising the peptide tag and between the peptide tag and the molecule comprising at least one binding moiety.

[0061] The terms "fragmentation", "digestion", "proteolysis", "proteolytic cleavage" "enzymatic cleavage", "non enzymatic cleavage" and "cleavage" as used herein is to be interpreted in the most inclusive manner and refers to all methods upon which the repetitive peptide sequences comprised in the peptide tag can be released. Such as release by using protease (enzyme) into peptides that can be later detected by LC-MS, or fragmentation where a peptide is broken up into its components (fragmentions) in the MS. Standard process during LC-MS acquisition.The term "sample" as used herein, is to be interpreted inclusively. That is, this word is intended to include all types of materials in any condition or volume that are suitable for MS detection.

[0062] In one aspect, present invention also relates to the following embodiments or items

[0063] The following embodiments are considered non-limiting to the invention

[0064] 1. Use of molecules comprising at least one binding moiety in a sample for MS-detection, wherein the binding moiety is linked and / or fused and / or conjugated with a peptide tag, wherein the peptide tag has a sequence which is uniquely identifiable by standard MS-detection methods. 2. Use according to embodiment 1, wherein the peptide tag is a peptide sequence native to the sample.

[0065] 3. Use according to embodiment 1-2, wherein the peptide tag is a peptide sequence not native to the sample.

[0066] 4. Use according to any one of the preceding embodiments, wherein the sequence length of the peptide tag is selected from 1-5 amino acids, 5-10 amino acids, 10-15 amino acids, 15-20 amino acids, 20-25 amino acids, 25-30 amino acids, 30-35 amino acids, preferably 6-25 amino acids. 5. Use according to any one of the preceding embodiments, wherein a plurality of peptide tagsequences are serially linked together, in repetitive sequences, to amplify signal detection. 6. Use according to any one of the preceding embodiments, wherein a plurality of peptide tagsequences are serially linked together, in repetitive sequences and wherein the links optionally further comprise one or more amino acids not part of the peptide tag sequence.

[0067] 7. Use according to any one of the embodiments, wherein the tag peptide sequences which are serially linked together are multiple copies of the peptide sequence e.g., 1 copy, 2 copies, 3 copies, 4 copies, 5 copies, 6 copies, 7 copies, 8 copies, 9 copies, 10 copies, 11 copies, 12 copies, 13 copies, 14 copies, 15 copies, 16 copies, 17 copies, 18 copies, 19 copies, 20 copies, 21 copies, 22 copies, 23 copies, 24 copies, 25 copies, 26 copies, 27 copies, 28 copies, 29 copies, 30 copies, 31 copies, 32 copies, 33 copies, 34 copies, 35 copies, 36 copies, 37 copies, 38 copies, 39 copies, 40 copies, 41 copies, 42 copies, 43 copies, 44 copies, 45 copies, 46 copies, 47 copies, 48 copies, 49 copies, 50 copies, 55 copies, 60 copies, 65 copies, 70 copies, 75 copies, 80 copies, 85 copies, 90 copies, 95 copies, 100 copies, 105 copies, 110 copies, 115 copies, 120 copies.

[0068] 8. Use according to any one of the embodiments, wherein the serially linked peptide tag sequences may be multiple copies of the same peptide sequence such as 1 copy, such as 2 copies, such as 3 copies, such as 4 copies, such as 5 copies, such as 6 copies, such as 7 copies, such as 8 copies, such as 9 copies, such as 10 copies, such as 11 copies, such as 12 copies, such as 13 copies, such as 14 copies, such as 15 copies, such as 16 copies, such as 17 copies, such as 18 copies, such as 19 copies, such as 20 copies, such as 21 copies, such as 22 copies, such as 23 copies, such as 24 copies, such as 25 copies, such as 26 copies, such as 27 copies, such as 28 copies, such as 29 copies, such as 30copies, such as 31 copies, such as 32 copies, such as 33 copies, such as 34 copies, such as 35 copies, such as 36 copies, such as 37 copies, such as 38 copies, such as 39 copies, such as 40 copies, such as 41 copies, such as 42 copies, such as 43 copies, such as 44 copies, such as 45 copies, such as 46 copies, such as 47 copies, such as 48 copies, such as 49 copies, such as 50 copies, such as 55 copies, such as 60 copies, such as 65 copies, such as 70 copies, such as 75 copies, such as 80 copies, such as 85 copies, such as 90 copies, such as 95 copies, such as 100 copies, such as 105 copies, such as 110 copies, such as 115 copies, such as 120 copies or a variation thereof.

[0069] 9. Use according to any one of the embodiments, wherein the serially linked peptide tag sequences may be between 2 and 32 copies of the same peptide sequence.

[0070] 10. Use according to any one of the preceding embodiments wherein a first end of a first peptide tag is linked and / or fused and / or conjugated to one or more sites on the molecule comprising the binding moiety.

[0071] 11. Use according to any one of the preceding embodiments wherein a first end of a first peptide tag is linked and / or fused and / or conjugated to one or more heavy and / or light chains of the molecule comprising the binding moiety.

[0072] 12. Use according to any one of the preceding embodiments wherein a first end of the first peptide tag is linked and / or fused and / or conjugated to each heavy chain and / or light chain of the binding moiety.

[0073] 13. Use according to any one of the preceding embodiments, wherein the linking and / or fusing and / or conjugation of the first end of the first peptide tag is at the N-terminal and / or C-terminal end of the binding moiety.

[0074] 14. Use according to any one of the preceding embodiments, wherein the linking and / or fusing and / or conjugation of the first end of the first peptide tag is at the N-terminal and / or C-terminal end of the heavy chain of the binding moiety.

[0075] 15. Use according to any one of the preceding embodiments, wherein the linking and / or fusing and / or conjugation of the first end of the first peptide tag is at the N-terminal end of the heavy chain of the binding moiety.

[0076] 16. Use according to any one of the preceding embodiments, wherein the linking and / or fusing and / or conjugation of the first end of the first peptide tag is at the C-terminal end of the heavy chain of the binding moiety.

[0077] 17. Use according to any one of the preceding embodiments, wherein the MS-detected signal of the peptide tag is directly proportional to the quantity of the binding moiety-directed-protein detected in the sample.18. Use according to any of the preceding embodiments, wherein the linking and / or fusing and / or conjugation to the binding moiety and / or the link between the peptide tags are amenable to enzymatic cleavage during enzymatic digestion treatment or released by other means.

[0078] 19. Use according to any of the preceding embodiments, wherein the peptide cleavage treatment is denaturation and or cleavage by a protease and / or a chemical agent and / or any other physical means.

[0079] 20. Use according to any of the preceding embodiments, wherein the sample is a single cell, derived from mammals, plants, eukaryotes and prokaryotes.

[0080] 21. Use according to any of the preceding embodiments, wherein the sample is a plurality of cells, derived from mammals, plants, eukaryotes and prokaryotes.

[0081] 22. Use according to any of the preceding embodiments, wherein the sample is biofluids, cell lysates, tissue biopsies, preserved clinical specimens, or fresh material derived from mammals, plants, eukaryotes and prokaryotes.

[0082] 23. Use according to any of the preceding embodiments, wherein beyond the signal amplification peptide tag a fluorophore, and / or chromogen, and / or metal particle and / or other imaging-based detection agent is linked, conjugated or by other means attached to the binding.

[0083] 24. Use according to any one of the preceding embodiments wherein the molecule comprising the binding moiety is an antibody or a single domain antibody or an antibody binding member, or a mini-bodies or a DARPin or an armadillo repeat protein or a bicyclic peptides.

[0084] 25. Use according to any one of the preceding embodiments, wherein the molecule comprising the binding moiety is an antibody, single domain antibody, antibody binding member, mini-body DARPin, armadillo repeat protein, bicyclic peptides, scFv-Fc, sdAb-Fc, nanobody-Fc, Fab, scFv, sdAb, nanobody, or another antibody fragment, minibody, triabody, tetrabody, dAbs-Fc, Fab, scFv, (SCFV)2, dAbs, VHH, diabody, sdAb, nanobody, scDb and taF.

[0085] 26. Use according to any one of the preceding embodiments, wherein the molecule comprising the binding moiety is an antibody, or antibody binding member.

[0086] 27. Use according to any one of the preceding embodiments, wherein the molecule comprising the binding moiety is an antibody with binding affinity towards HER2 or PDL1.

[0087] 28. A molecule comprising at least one binding moiety, wherein the binding moiety is linked and / or fused and / or conjugated with a peptide tag, wherein the peptide tag has a sequence which is uniquely identifiable by standard MS-detection methods.

[0088] 29. The molecule according to embodiment 28, wherein the peptide tag is defined according to anyone of embodiments 1-19.30. A method for detecting the number of e.g., proteins-of-interest in a sample, the method comprising

[0089] a) providing a biological sample that comprises one or more proteins of interest,

[0090] b) contacting the sample of a) with a molecule comprising at least one binding moiety with affinity towards the protein of interest that is linked and / or fused and / or conjugated to a peptide-tag as defined in any of embodiments 1-24

[0091] c) subjecting the mixture of a) and b), to a fragmentation step, whereby the conjugated peptides are digested,

[0092] d) detecting the digested peptides by any suitable technique based on mass-spectrometry, wherein the signal obtained from the peptide sequence correlates to the quantity of protein-of- interest in the sample a).

[0093] 29. The method according to embodiment 30 wherein the fragmentation step c) comprises adding an enzyme such as trypsin to the mixture of a) and b).

[0094] 30. The method according to embodiment 30 wherein the fragmentation step c) comprises a physical fragmentation such as collision-induced-, PSD-MALDI-, higher energy C-trap-, electron transfer-, electron capture-, in source-, post-source and ultraviolet photo-dissociation to the mixture of a) and b).Examples

[0095] METHODS

[0096] The present invention is further exemplified, but not limited by, the following examples that illustrate the use of the unique identifiable peptide tags and other examples anywhere in the specification is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified form. Likewise, the invention is not limited to any particular preferred embodiments described herein. Indeed, many modifications and variations of the invention may be apparent to those skilled in the art upon reading this specification and can be made without departing from its spirit and scope. The invention is therefore to be limited only by the terms of the appended claims, along with the full scope of equivalents to which the claims are entitled.

[0097] Unless otherwise stated:

[0098] • operations were generally carried out at ambient temperature, that is typically between 18 and 26°C and without exclusion of air unless otherwise stated, or a person skilled in the art would otherwise work under an inert atmosphere;

[0099] • cell lines were kept at 37°C in a humidified incubator with 5% CO2

[0100] • FACS were performed using MACSQuant Analyzer 16 flow cytometer (Miltenyi) and a Sony Sorter MA900

[0101] • Mass spectra were obtained using an Orbitrap Eclipse mass spectrometer connected to FAIMS Pro operating at -45 V with a Ultimate 3000 RSLCnano system with pPAC NEO Trapping Column, low load pre-column (Thermo Scientific) and pPAC NEO HPLC Column 50 cm (Thermo Scientific).

[0102] Abbreviations

[0103] As used herein, the symbols and conventions are consistent with those used in contemporary scientific literature. Specifically, the following abbreviations are used in the text:

[0104] Antibody components, reagents and buffers:

[0105] HC, Heavy Chain; LC, Light Chain; PDL1, Programmed Death-Ligand 1; HER2, Human Epidermal Growth Factor Receptor 2; mAb, Monoclonal Antibody; PBS, Phosphate-Buffered Saline; DPBS, Dulbecco's Phosphate-Buffered Saline; TEAB, Triethylammonium Bicarbonate; TFA, Trifluoroacetic Acid; TFE, Trifluoroethanol; DTT, Dithiothreitol; SDC, Sodium Deoxycholate; TCEP, Tris(2-carboxyethyl)phosphine; CAA, Chloroacetamide.

[0106] Cell lines and cell culture:

[0107] HEK, Human Embryonic Kidney; FBS, Fetal Bovine Serum; WT, Wild-Type.

[0108] Molecular techniques

[0109] PCR, Polymerase Chain Reaction; HIFI, High-Fidelity PCR; IPTG, Isopropyl p-D-l-thiogalactopyranoside; ELISA, Enzyme-Linked Immunosorbent Assay; FACS, Fluorescence-Activated Cell Sorting; SDS-PAGE, Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis; MS, Mass Spectrometry, DIA, Data-independent Acquisition, PRM, Parallel Reaction Monitoring; CMV, Cytomegalovirus; IgG,Immunoglobulin G; MCS, Multiple Cloning Site; ORI, Origin of Replication; AmpR, Ampicillin Resistance; EC50, Half Maximal Effective Concentration

[0110] EXAMPLE 1: Design and production of a tagged antibody

[0111] In this experiment antibodies with peptide tags are expressed, purified, and characterized, they are exhibiting high specificity, stability, and compatibility with immunodetection and mass spectrometrybased applications.

[0112] Antibody design and cloning

[0113] Antibodies targeting PDL1 or HER2 were attached to peptide tags comprising 2-32 copies of the desired peptide sequence. PDL1 antibodies were attached to peptide tags wherein the repetitive peptide sequence had the following amino acid sequence TASEFDSAIAQDK, SEQ ID 4 and HER2 antibodies were attached to peptide tags wherein the repetitive peptide sequence had the following amino acid sequence LTILEELR SEQ ID 8, using PCR and HIFI technologies. DNA encoding the peptide tags of interest were purchased (Genwiz) The peptide tag was anchoraged with a flexible GGGGS linker (10-mer) to the heavy chain (HC). HC and light chain (LC) were synthesized and cloned into a mammalian expression vector containing the human IgGl constant region and kappa / lambda constant region for HC and LC, respectively. The expression constructs were designed with the CMV promoter for high expression in HEK293 cells, a human IgG secretion signal sequence (HSA sequence) to facilitate secretion, Carbenicillin resistance gene for stable selection and the peptide-tag located either N- or C-terminal in the variable region from the HC. PDL1 was tagged with the peptide tag TASEFDSAIAQDK (nucleotide SEQ ID 2 and amino acid SEQ ID 4) at the N -terminal, with between 1 and 16 serially repeated peptide sequences per HC (nucleotide SEQ ID 1 and amino acid SEQ ID 3), using the following format HSA signal:: (peptide tag)n:: GGGGS linker::PDLl variable heavy sequence:: CD20. HER2 was tagged with the peptide sequence LTILEELR (nucleotide SEQ ID 6 and amino acid SEQ ID 8) at the C-terminal, the peptide tag comprising between 8 and 16 serially repeated peptide sequences per HC (nucleotide SEQ ID 5 and amino acid SEQ ID 7), using the following format HSA sequence:: HER2 variable heavy sequence:: CD20::GGGGS linker:: (peptide tag)n.

[0114] The plasmids encoding the HC and LC genes were sequence-verified and prepared using a midiprep plasmid purification kit.

[0115] Peptide tagged antibody nucleotide and amino acid sequences are disclosed in table 1(peptid

[0116] e tag

[0117] se GGGS

[0118] Table 1 HSA signal quen Antibody (variable heavy) sequence CD20 antibody sequence

[0119] ce)nlinker

[0120] n=l-16

[0121] G CTTCCACCAAG G G ACCCTCTGTGTTCCCTCTG G CTCCTTCCAG CAAGTCCACA GAGGTGCAGCTGGTGGAATCTGGCGG TCTGGTGGTACcgccgctctgggctgcctcgtgaaggactacttccccgagcccgtgacagtgtcct AG G ATTG GTTCAG CCTG GCGG CTCTCT

[0122] ggaactctggcgctctgaccagcggagtgcacaccttccctgctgtgctgcagtcctccggcctgtactc GAGACTGTCTTGTGCCGCTTCCGGCTT

[0123] cctgtccagcgtcgtgactgtgccctccagctctctgggcacccagacctacatctgcaacgtgaaccac GGTG TACCTTCTCCG ACTCCTG G ATCCACTG G ATGAAATG aagccctccaacaccaaggtggacaagaaggtggaacccaagtcctgcgacaagacccacacctgtc Peptide accgcga GGGG GTCCGACAGGCACCTGGCAAAGGACT

[0124] GGTCACCTT ccccttgtcctgcccctgaactgctgggcggacccagcgtgttcctgttccccccaaagcccaaggatac tagged PDL1 gcgaatt CGGC GGAATGGGTCGCCTGGATCTCTCCTTA

[0125] TATCTCCCT cctgatgatctcccggacccccgaagtgacctgcgtggtggtggatgtgtcccacgaggaccctgaagt nucleotide tgatagc TCCG CG G CGG CTCTACCTACTACG CCG ACTC

[0126] GCTGTTCCT gaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcctagagaggaacag sequence (N- gcgattg GCGG TGTGAAGGGCAGATTCACCATCTCCGC

[0127] GTTCTCCAG tacaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagag terminal cgcagg GGGA CGACACCTCCAAGAACACCGCCTACCT

[0128] CGCCTACTC tacaagtgcaaggtgtccaacaaggccctgcctgcccccatcgaaaagaccatctccaaggccaaggg tagged) ataaa GGAA GCAGATGAACTCCCTGAGAGCCGAGG

[0129] T ccagccccgggaaccccaggtgtacacactgccccctagcagggacgagctgaccaagaaccaggtg GC ACACCG CCGTGTACTACTGTG CTAG AA

[0130] tccctgacatgcctcgtgaaaggcttctacccctccgatatcgccgtggaatgggagtccaacggccag GGCATTGGCCAGGCGGCTTCGATTATT

[0131] cctgagaacaactacaagaccaccccccctgtgctggactccgacggctcattcttcctgtacagcaag GGGGCCAGGGAACACTGGTCACCGTG

[0132] ctgacagtggacaagtcccggtggcagcagggcaacgtgttctcctgctccgtgatgcacgaggccctg TCCTCT

[0133] cacaaccactatacccagaagtccctgtccctgagccccggcaagtga

[0134] Peptide ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV EVQLVESGGGLVQPGGSLRLSCAASGFT

[0135] tagged PDL1 LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP TASEFD GGGG FSDSWIHWVRQAPGKGLEWVAWISPY

[0136] amino acid MKWVTFISL APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH SAIAQD SGGG G GSTYYADSV KG R FTISADTSK NTAYLQ

[0137] sequence (N- LFLFSSAYS NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG K GS MNSLRAEDTAVYYCARRHWPGGFDYW

[0138] terminal QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV GQGTLVTVSS

[0139] tagged) LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

[0140]

[0141] (peptid

[0142] Table 1 HSA e tag GGGS

[0143] sequen Antibody (variable heavy) sequence CD20 antibody sequence

[0144] (continued) signal linker

[0145] ce)„

[0146] n=i-i6

[0147] GCCAGTACTAAAGGTCCTTCGGTCTTCCCCCTGGCTCCCTCCAGCAAGTCAACT TCCGGAGGTACTGCCGCACTGGGGTGCCTGGTTAAGGACTATTTTCCAGAGCC AGTAACAGTGAGTTGGAATAGCGGTGCTCTCACCTCCGGAGTGCATACATTTC CAGCG GTATTG CAGTCTTCG GGG CTATATAG CCTG AGCTCAGTG GTTACTGTG CCATCCTCCTCTCTCGGGACTCAAACTTACATATGCAATGTGAATCACAAGCCC

[0148] gaggtgcagcttgtcgaatctggcgggggtcttgtcc TCAAATACCAAGGTCGACAAGAAGGTTGAGCCCAAAAGTTGTGATAAGACAC ATGAAAT GGTG aacccggtggctctttgcgcctgtcctgcgcagcgtc ACACCTGTCCACCATGCCCGGCCCCCGAGCTGCTGGGCGGCCCTTCAGTCTTTC

[0149] Peptide tagged GGGTCAC GGG cggctttaacatcaaggatacgtacattcactgggtg

[0150] CTCAC TGTTCCCTCCAAAACCGAAAGACACGCTAATGATCAGCAGGACTCCGGAGGTG HER2 CTTTATCT GCGG agacaggctccaggaaagggattagagtgggttgc

[0151] CATCC ACCTGTGTGGTGGTGGACGTGAGTCACGAAGATCCGGAGGTTAAGTTCAATT

[0152] nucleotide CCCTGCT CTCC ccgcatttaccctacaaatggatatacaaggtatgcc

[0153] TGGAG GGTACGTGGACGGCGTTGAAGTCCACAACGCTAAGACAAAACCTAGAGAGGA

[0154] sequence (C- GTTCCTG GGCG gattccgttaaaggacggtttaccatttctgctgatac

[0155] GAGTT ACAGTATAACAGTACCTACAG AGTG GTCAG CGTCTTAACCGTACTTCATCAG G

[0156] terminal TTCTCCA GGG aagcaagaacacagcctaccttcagatgaacagtct

[0157] AAGA ACTG G CTCAACG G G AAAG AATATAAATGTAAAGTCTCTAACAAGG CCTTGCCA

[0158] tagged) GCGCCTA GAGG cagggcagaagacactgcagtctactattgctctcg GCACCTATCGAGAAAACTATAAGCAAGGCGAAGGGGCAGCCCCGTGAGCCTC CTCT AAGC atggggaggggacggcttctatgccatggattactg AGGTGTACACACTCCCCCCTTCACGGGAGGAGATGACCAAAAACCAGGTGTCG

[0159] gggtcaggggacgctggtgaccgtgagctcc

[0160] CTGACCTGTCTGGTGAAGGGATTCTATCCTTCTGATATCGCTGTTGAGTGGGA AAGCAATGGCCAACCCGAAAATAACTACAAGACCACACCCCCCGTGCTGGACA GCGATGGCTCCTTCTTCCTGTACTCAAAACTCACCGTCGACAAATCCCGGTGGC AACAG G GCAATGTATTTTCTTG CAG CGTAATGC ACG AAG CCCTCCATAACCATT ACACGCAGAAGTCTTTAAG 1 1 1 G 1 CACCTGGCAAA

[0161] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV

[0162] Peptide tagged EVQLVESGGGLVQPGGSLRLSCAASGFNI LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP HER2 amino MKWVTFI GGG KDTYIHWVRQAPGKGLEWVARIYPTNGY

[0163] LTILEEL APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH

[0164] acid sequence SLLFLFSSA GSGG TRYADSVKGRFTISADTSKNTAYLQMNSL

[0165] R NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG

[0166] (C-terminal YS GGS RAE DTAVYYCSRWG GDG FYAM DYWGQ QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP

[0167] tagged) GTLVTVSS

[0168] VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0169]

[0170] Transient transfection into HEK cells and purification

[0171] HEK293F cells (serum-free) were cultured in HEK-optimized FreeStyle 293 Expression Medium at 37°C, 5% C02, and 120 rpm shaking. Transient transfection and purification were performed using the following protocol:

[0172] 1. HEK293F cells were seeded at 1 x 106cells / mL in fresh media.

[0173] 2. PEI-mediated transfection was carried out at a DNA:PEI ratio of 1:2 (w / w).

[0174] 3. Plasmids encoding HC and LC (3:2 ratio) were diluted in DMEM and mixed with PEI before adding to the cells.

[0175] 4. The cells were incubated for 7 days,

[0176] 5. and supernatants were collected by centrifugation at 300 x g for 10 minutes, followed 3000 x g for 10 minutes, and 0.22 pm filtered to remove cell debris.

[0177] 6. The supernatant was loaded onto a pre-equilibrated Protein A Sepharose column at 4°C. 7. The column was washed with sodium phosphate binding buffer (pH 7.3).

[0178] 8. The antibody was eluted with 0.1 M Tris buffer (pH 3) and immediately neutralized with Tris- HCI (pH 8.0).

[0179] 9. The eluate was buffer exchanged into PBS using Merck Amicon filters (30kDa MWCO) Purity was assessed by SDS-PAGE (4-20% Mini-PROTEAN® TGX™ Precast Protein Gels, Bio-Rad) in the presence and absence of 25 mM DTT and visualized through Coomassie Brilliant Blue staining.

[0180] Precision Plus Protein Dual Color Standards (Bio-Rad, 1610374) was included as reference. Purified antibodies were tested in non-reducing conditions, where they exhibited an expected molecular weight of approximately 150-185 kDa (dependent on tag length). Under reducing conditions, heavy and light chains migrated at ~50-85 kDa and ~25 kDa, respectively.

[0181] Stability studies, performed by SDS-PAGE, showed that the tagged antibodies remained structurally stable when stored at -20°C for 1 year, with no detectable aggregation compared to freshly produced antibodies

[0182] EXAMPLE 2: Binding characterization of the peptide tagged antibodies (Fig. 1 and Fig.2)

[0183] In this experiment we demonstrate that inclusion of 16 uniquely identifiable peptide tags (non-human peptides) per HC does not interfere with the intended binding capabilities of the antibody.

[0184] Human cell lines Raji WT, Raji PDL1+, HT29 and DU-145 were maintained in complete RPMI (RPMI 1640 Sigma-Aldrich) supplemented with 10% FBS (Gibco), 1% L-glutamine and 1% penicillin-streptomycin (Sigma-Aldrich). All cell lines were kept at 37°C in a humidified incubator with 5% CO2.

[0185] PDL1 tagged antibodies and HER2-tagged antibodies, were labelled according to manufacturer protocol with Zenon™ Human IgG Labeling Kit (Thermo Fisher Scientific, Z25402) and (Thermo Fisher Scientific, Z25408), respectively. Raji WT, Raji PD-L1+, HT29 and DU-145 were incubated for 10 min on ice with labelled antibodies (0-20 pg / mL), washed and fixed with fixation buffer (eBioscience™, 88-8824-00) prior to flow cytometric analysis (MACSQuant, Miltenyi). Data from three independent assays using cells from different passages was analyzed in Prism 10.3.1 (GraphPad Software) where data was fitted and EC50 values were calculated using the inbuilt nonlinear regression curve fit: log(agonist) vs. response - variable slope model. Statistical differences were calculated using one-way ANOVA using Geisser-Greenhouse correction and Tukey's correction for multiple comparisons (n=3).Analysis of the cell binding assay show that peptide-tagged antibodies bind to their target in a comparable way to a non-tagged (IgG control) antibody

[0186] EXAMPLE 3: Cell sorting of the peptide tagged antibodies (Fig. 1)

[0187] Human cell line DU-145 (PDL1+ / HER2+) was maintained in complete RPMI (RPMI 1640 Thermo Fisher Scientific, 42401018) supplemented with 10% FBS (Gibco), 1% L-glutamine and 1% penicillinstreptomycin (Sigma-Aldrich). The cell line was kept at 37°C in a humidified incubator with 5% CO2. Prior to FACS, DU-145 cells were washed in DPBS, and tested antibodies were labelled according to manufacturer protocol with either Zenon™ Human IgG Labeling Kit (Thermo Fisher Scientific, Z25402) or (Thermo Fisher Scientific, Z25408). Following this, l*106cells were incubated with labelled antibodies for 10 min on ice. Cells were then washed in DBPS to eliminate excess antibody before FACS sorting using a 130 pm sorting chip (Sony Sorter MA900). Doublets were excluded prior to sorting based on forward scatter (FSC)-H versus FSC-A, and dead cells were identified and excluded based on FSC versus back scatter (BSC). The PDL1 and HER2 populations were gated using unstained controls as references. Cells were sorted in a 384 well plate for digestion before MS.

[0188] In this experiment we show that the peptide tagged antibodies were capable of binding a single cell.

[0189] EXAMPLE 4: Identifying peptide tags on single cells (Fig. 4)

[0190] DU-145 (PDL1+ / HER2+) cells were stained with PDL1 antibodies with peptide tags with either 0, 2, 16 or 32 repetitive and serially linked repeats or HER2 antibodies with peptide tags with either 016 or 32 repetitive and serially linked repeats attached to either their N- or C-terminal. Only PDL1, HER2 or PDL1 and HER2 positive cells were sorted. The sorted single cells with bound peptide tagged antibodies were digested in a 384 Eppendorf™ twin. tec™ PCR Plates LoBind™ containing luL 20 % TFE (Sigma-Aldrich, 91683) with 80 mM TEAB (thermo scientific, 90114). The plate was snap frozen on dry ice and boiled to 96°C for 5 min followed by a second freeze cycle to -80 °C. 1 pL of 100 mM TEAB containing 2 ng Trypsin Platinum (VA9000, Promega, USA) was added to each well and the plate was incubated overnight. The digestion was stopped with 1 pL 1 %TFA. Fortargeted-MS experiments, isotope labelled peptides were added within the 1 % TFA. Isotope labelled peptides were added at a concentration of 1.56 amol / cell (Pierce® Peptide Retention Time Calibration Mixture PN 88320, 0.5pmol / pL, 50 pl). Samples were subjected to liquid chromatography coupled to mass spectrometry. A Gradient was built starting at 750 nL / min in 99 % Solvent A (1% Formic Acid). Solvent B (80% Acetonitrile, 1% Formic Acid). The gradient was increased to 8 % for 0.2 min followed by a drop in flow to 200 nL / min and a linear gradient to 24% Solvent B over 2.8min. Solvent B was increased to 48 % until minute 8 followed by 99 % B for 6.55 min. Column-equilibration was performed over 2.85 min and the next precolumn loading at 1 % B with a flowrate of 750 nL / min. Data was acquired in DIA and a DIA combined with targeted Acquisition. MSI DIA acquisitions were performed at 120k resolution (AGC target of 300 %, max injection time (IT) 246ms). MS2 scans were inserted (HCD of 26%, resolution 120k, 246 ms max IT, AGC target 1000 %) with 6 isolation windows covering 400 - 800 m / z. Targeted MS2 scans operated at 240k resolution with a max IT of 504 ms. Precursors isolation was performed at 5.7 or 6.7 m / z window. PRM and DIA scans were with a 0.7 min retention time window for the PRM.

[0191] No signal could be observed for the untagged PDL1 antibodies or HER2 antibodies. The PDL1 antibody could be identified with a peptide tag comprising 32 repetitive peptide sequences whereas the HER2antibody could be identified with a peptide tag comprising 16 repetitive peptide sequences. Antibodies with peptide tags comprising fewer repetitive peptide sequences could not be identified via MS. By correlating the MS signal of the sorted cells with the gold-standard fluorescence-based readout a positive Pearson correlation of 0.9 and 0.92 could be observed. These results could be replicated with an untargeted DIA method. Furthermore, the absolute quantification of the tag-peptides allowed for a calculation of antibody molecules that were bound to each cell; for the PDL1 antibody a median of 43173 and for the HER2 antibody a median of 7130. Besides the identification of both antibodies via the repetitive peptide tags, Till non-tagged proteins could be identified on average.

[0192] EXAMPLE 5: Multiplexing with peptide tagged PDL1 antibody and peptide tagged HER2 antibody (Fig.

[0193] 5)

[0194] Cells were stained, sorted and processed as described in Experiment 3 and 4. Except that cells were only stained with a combination of the PDL1 antibody tagged with the peptide tag comprising 32 repetitive peptide sequences and the HER2 antibody tagged with the peptide tag comprising 16 repetitive peptide sequences. Only PDL1 and HER2 double positive cells were sorted. Single cells, 2 cells and 4 cells were sorted in each well.

[0195] A high degree of correlation could be observed between the gold-standard fluorescent based (FACs) quantification and the MS based quantification on the double labelled over the range of 1-4 cells. A person correlation of 0.8 and 0.59 could be observed for the PDL1 antibody and the HER2 antibody, respectively. Furthermore, at median of 3007, 3502 and 3966 additional proteins could be identified in 1, 2 and 4 cells.

[0196] EXAMPLE 6: Increased number of repetitive peptide tags on the PDL1 antibody increases sensitivity (Fig. 3)

[0197] PDL1 antibodies tagged with a peptide tag comprising either 2 or 32 repetitive peptide sequences were digested with trypsin. The antibodies were heated to 95°C for 10 min, reduced in 2% SDC, lx PBS, 10 mM TCEP for 30min at 37°C, alcylation at RT for 30 min in 40 mM CAA after which the samples were diluted to 0.1 % SDC. Trypsin was added in a 1:25 ratio. The sample was digested at 37°C ON and the reaction was quenched to a final concentration of 0.5 %TFA. The digest was desalted using the SOLAp™ SPE Plates (Thermo Scientific™). The antibody-peptide-tag digest was diluted in 100 pg / pL Pierce™ HeLa Protein Digest Standard. Isotope labeled peptides (Pierce® Peptide Retention Time Calibration Mixture PN 88320, 0.5pmol / pL, 50 pl) of the peptide tags were added at a concentration of 1.56 amol / pL.

[0198] At the same concentration of antibody, the antibody tagged with the peptide tag comprising the 32 repetitive peptide sequences had a higher signal over the antibody tagged with the peptide tag comprising 2 repetitive peptide sequences. For the peptide tags and antibody in question we observe a decreased LOQ with increasing number of repetitive peptide sequences in the peptide tag; 32-repetitive peptide sequences had an LOQ of 0.049 % of the starting antibody dilution and the 2-repetitive peptide sequences displayed a LOQ of 0.415 %.

[0199] EXAMPLE 7:

[0200] A monoclonal antibody (mAb) specific to a low-abundance intracellular protein, such as a transcription factor (e.g., MYC) is generated and engineered to include a peptide tag comprising between 4 and 12tandemly repeated peptides sequences (AbTag). The AbTag is designed such that each peptide repeat contains a sequence that is uniquely identifiable by LC-MS / MS and is cleavable by trypsin digestion, thereby enabling release of reporter peptides during standard bottom-up proteomics sample preparation.

[0201] Cells expressing the target protein are fixed and permeabilized using a standard protocol (e.g., paraformaldehyde fixation followed by Triton X-100 or Methanol permeabilization), followed by incubation with under conditions permitting specific binding (e.g., overnight at 4°C in PBS with 1% BSA).

[0202] Following binding, the cells are subjected to proteolytic digestion (e.g., trypsin digestion), resulting in the simultaneous generation of 1) endogenous peptides derived from the cellular proteome, and 2) AbTag-derived peptide reporters released from the AbTag molecule.

[0203] The resulting peptide mixture is analyzed by LC-MS / MS. The AbTag-derived peptides are detected based on their unique mass-to-charge ratios and fragmentation patterns, and the signal intensity is proportional to the number of peptide repeats and the abundance of the bound target protein. This approach enables simultaneous detection of the target protein (e.g. MYC) and global proteome profiling within a single bottom-up proteomics workflow, while providing signal amplification through tandem peptide repeats, thereby facilitating detection of low-abundance intracellular proteins. As portrayed in Example 5, multiple AbTags can be combined at the same time, providing multiplexed AbTag-derived readouts of multiple protein targets of interest alongside global proteome readouts, using a single sample preparation procedure and a single LC-MS run.

Claims

CLAIMS1. A peptide tag for Mass Spectrometry (MS) detection of an analyte in a sample, the peptide tag having a first and a second end, the peptide tag comprising two or more serially linked repetitive peptide sequences,wherein the first end of the peptide tag may be attached to a molecule comprising at least one binding moiety, andwherein the repetitive peptide sequences are cleavable and linked to each other,wherein the sequence length of each of the serially linked repetitive peptide sequences is between 1 and 35 amino acids such as 1-5 amino acids, 5-10 amino acids, 10-15 amino acids, 15-20 amino acids, 20-25 amino acids, 25-30 amino acids, 30-35 amino acids, preferably 6-25 amino acids, and wherein the peptide tag associated signal obtained from the MS spectra is directly proportional to the absolute number of repetitive peptide sequences in the peptide tag, andwhereby correlation to the total number of analyte molecules in the sample can be made.

2. The peptide tag according to claim 1, wherein the attachment between the first end of the peptide tag and the molecule comprising at least one binding moiety is sensitive to fragmentation and / or proteolysis,such as by enzymatic cleavage, chemical cleavage, physical fragmentation, electrochemical and catalytic methods, andwherein the molecule comprising at least one binding moiety is an antibody, or single domain antibody, or antibody binding member or minibinder , or DARPin, or armadillo repeat protein, or bicyclic peptides, or triabody, or tetrabody, or dAbs-Fc, or Fab, or scFv, or (scFv)2, or dAbs, VHH, or diabody, or scDb or taF.

3. The peptide tag according to claims 1-2, wherein the molecule comprising at least one binding moiety may comprise additional detection agents for detection by imaging such as a fluorophore, and / or chromogen, and / or metal particle.

4. The peptide tag according to any of the preceding claims, wherein one or more of a peptide tag may be attached to the molecule comprising at least one binding moiety.

5. The peptide tag according to any of the preceding claims, wherein the cleavable link between the repetitive peptide sequences is sensitive to proteolytic cleavage and / or fragmentation.

6. The peptide tag according to any of the preceding claims, wherein the repetitive peptide sequence is selected to have a mass-to-charge ratio (m / z) that is distinct from the m / z values of endogenous peptides present in the sample.

7. The peptide tag according to any of the preceding claims, wherein the amino acid composition of the repetitive peptide sequences is amenable to MS ionization.

8. The peptide tag according to any of the preceding claims, wherein the number of serially linked repetitive sequences are an integer between 2 and 120.

9. The peptide tag according to any of the preceding claims, wherein the number of repetitive peptide sequences is between 2 and 8 repetitive peptide sequences.

10. The peptide tag according to any of the preceding claims, wherein the number of repetitive peptide sequences is between 8 and 32 repetitive peptide sequences.

11. The peptide tag according to any of the preceding claims, wherein the number of repetitive peptide sequences is at least 34 repetitive peptide sequences.

12. The peptide tag according to any of the preceding claims, wherein the repetitive peptide sequences are selected from SEQ ID 4 or SEQ ID 8.

13. The peptide tag according to any one of the preceding claims, wherein the molecule comprising at least one binding moiety is an antibody with binding affinity towards HER2 or PDL1, and wherein the repetitive peptide sequence is selected from SEQ ID 4, or SEQ ID 8, andwherein the number of serially linked repetitive peptide sequences in the peptide tag is between 2 and 32, andwherein the first end of one or two peptide tags is attached to the antibody, andwherein the attachment is by way of linking and / or fusing and / or conjugation, andwherein the linking and / or fusing and / or conjugation of the one or two peptide tags are to the C- or N-terminal of the antibody chains, andwherein the peptide tag associated MS detection signal is directly proportional to the total number of repetitive peptide sequences on the sample, andwhereby the correlation to the total number of HER2 or PDL1 analyte molecules can be made.

14. Use of a peptide tag according to claim 1 to 13, to amplify an analyte signal in a sample undergoing analysis using MS detection.

15. Use of a peptide tag according to claim 14 to determine the total number of analyte molecules in a sample undergoing analysis using MS detection.

16. Use of a peptide tag according to claims 14-15, wherein the sample is a single cell, derived from mammals, plants, eukaryotes and / or prokaryotes.

17. Use of a peptide tag according to claim 14-16, wherein the sample is a plurality of cells, derived from mammals, plants, eukaryotes and / or prokaryotes.

18. Use of a peptide tag according to claim 14-17, wherein the sample is biofluids, cell lysates, tissue biopsies, preserved clinical specimens.

19. A method for detecting an analyte in a sample, particularly the total number of an analyte in a sample, the method comprisinga) providing a biological sample that comprises one or more analytes-of-interest,b) contacting the sample of a) with a peptide tag according to claims 1-13 wherein the molecule comprising at least one binding moiety attached to the peptide tag has affinity towards an analyte of interest in the sample,c) subjecting the mixture of a) and b), to a fragmentation and / or proteolytic step, whereby the conjugated peptides are released and / or digested,d) detecting the released and / or digested peptides by any MS based technique such as LC-MS / MS, MALDI-TOF, and wherein the signal obtained from the repetitive peptide sequences in the fragmented peptide tag correlates to the quantity of the analyte in the sample a).

20. The method according to claim 19 wherein the fragmentation and / or proteolytic step c) comprises adding an enzyme such as trypsin to the mixture of a) and b).

21. The method according to claims 19-20 wherein the fragmentation step c) comprises a physical fragmentation such as collision-induced-, PSD-MALDI-, higher energy C-trap-, electron transfer-, electron capture-, in source-, post-source and ultraviolet photo-dissociation, or any other peptide fragmentation method to the mixture of a) and b).

22. A kit of parts for MS detection of an analyte in a sample, the kit comprising:a) a peptide tag according to claims 5 to 12,b) a coupling reagent configured to attach the first end of the peptide tag to a molecule comprising at least one binding moiety,wherein the first end of the peptide tag comprises a reactive group configured to attach to the molecule comprising at least one binding moiety.

23. The kit according to claim 22, wherein the reactive group is compatible with click chemistry, such as an azide or alkyne.

24. An analyte detection system comprising:a) a fusion protein comprisinga peptide tag domain according to claims 1 to 13, anda moiety binding domain according to claims 2 and 3, andb) a nuclear acid molecule encoding the fusion protein of a), andwherein the system may be recombinantly expressed in a eukaryotic, prokaryotic, or cell-free expression system.

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