(Poly)tagged signal enhancer

Abstract

A first aspect of the present invention relates to the use of a (poly)label to generate a quantifiable signal for an analyte of interest in mass spectrometry, the (poly)label having structure (I). In a second aspect, the present invention relates to a process for modifying an analyte of interest to obtain an increased intensity signal in mass spectrometry. A third aspect of the present invention relates to a method for determining an analyte of interest by mass spectrometry. A fourth aspect of the present invention relates to a (poly)label having the structure of formula (Ia). In a fifth aspect, the present invention relates to a reaction product comprising a polypeptide and a (poly)label having the general structural formula (III).

Description

[Technical Field]

[0001] A first aspect of the present invention relates to the use of a (poly)label to generate a quantifiable signal for an analyte of interest in mass spectrometry, the (poly)label having structure (I). In a second aspect, the present invention relates to a process for modifying an analyte of interest to obtain an increased intensity signal in mass spectrometry. A third aspect of the present invention relates to a method for determining an analyte of interest by mass spectrometry. A fourth aspect of the present invention relates to a (poly)label having the structure of formula (I). In a fifth aspect, the present invention relates to a reaction product comprising a polypeptide and a (poly)label having the general structural formula (III). [Background technology]

[0002] Mass spectrometry (MS) is a widely used technique for the qualitative and quantitative analysis of chemicals ranging from small molecules to macromolecules. Generally, it is a highly sensitive and specific method, even capable of analyzing complex biological samples, such as environmental or clinical samples. However, the sensitivity of measurement remains an issue for some analytes, especially when analyzed from complex biological matrices. MS is often coupled with chromatographic techniques, particularly gas chromatography (GC) and liquid chromatography (LC). Here, molecules of interest are separated by chromatography and individually subjected to mass spectrometry. However, there remains a need to improve the sensitivity of MS analytical methods, especially for the analysis of analytes that are low in abundance or when scarce material is available (e.g., biopsy tissue).

[0003] To increase the signal intensity of analytes such as peptides for LC-MS analysis, two general strategies have been demonstrated in the literature: (1) The use of LC mobile phase additives such as dimethyl sulfoxide (DMSO) or ethylene glycol (Hahne et al. 2013). (2) Chemical derivatization to add a permanent positive charge and / or to enhance the hydrophobicity of the analyte to enhance electrospray ionization ( Mirzaei et al., 2006 ).

[0004] Regarding the second option, i.e., signal enhancement by derivatization, derivatization reagents such as quaternary ammonium salts, phosphonium salts, or pyridinium salts have been described for peptide analysis. However, the prior art has several drawbacks. For example, regarding mobile phase additives, it has been reported that adding up to 5% DMSO to LC solvents enhances electrospray ionization (ESI) of peptides, but the degree of ESI enhancement is sample-volume dependent and peptide- and instrument-specific (Hahne et al., 2013). Furthermore, continuous use of DMSO in LC solvents requires frequent cleaning of the front-end instrument optics, thereby reducing the robustness of LC-MS instruments and increasing downtime.

[0005] When considering derivatization to increase ESI of peptides, it should be noted that this primarily works on peptides that do not ionize sufficiently in their native form (Mirzaei et al., 2006). As outlined by Mirzaei et al., peptides larger than 500 Da and peptides containing cationic amino acids (histidine, lysine, arginine) do not benefit much from derivatization. This makes the actual benefit of derivatization for peptide analysis very limited, since essentially all peptides used for LC-MS quantification contain cationic amino acids (arginine or lysine from trypsin digestion) and are larger than 500 Da. Peptides with a mass of 500 Da, corresponding to roughly 4–5 amino acids in length, are not unique and therefore not useful for protein identification and quantification. Summary of the Invention

[0006] Therefore, the problem underlying the present invention was to provide means and methods for increasing analyte signals in mass spectrometry.

[0007] First Aspect—Use of (Poly)Labels to Generate a Quantifiable Signal for an Analyte of Interest A first aspect of the present invention relates to the use of a (poly)label to generate a quantifiable signal for an analyte of interest in mass spectrometry, the (poly)label having the structure (I): [ka] During the ceremony, m is 0 or 1; n is 0 or an integer selected from the range of 1 to 20; Q is absent or a linker unit; X is a reactive group or, if Q is absent, a hydrogen atom; Y is an amino acid-based linker unit having a side chain suitable for coupling to Z; Y 1 is a linker unit based on an amino acid having a side chain suitable for coupling to Z, the amino acid having a blocked carboxylic acid group or a blocked amino acid group; Z is a group consisting of: - a nucleoside comprising a nucleobase selected from the group consisting of: adenine, cytosine, thymine, guanine and uracil; -Structure Z 1 -Proline-Z 2 A tripeptide of the formula: 1 and Z 2 are independently alanine, glycine, an amino acid selected from the group consisting of valine, leucine, iso-leucine, and phenylalanine; and -Carbamate group -OC(=O)-NH-(CH2) z -N + (Z 3 )3 or -NH-C(=O)-O-(CH2) z -N + (Z 3) 3 [wherein z is an integer selected from the range of 1 to 10, Z 3 is a C1-C5 alkyl group.

[0008] In the context of the present invention, "(poly)label" means a label comprising one or more moieties selected from the group Z above, where if there is only one moiety it is a "label" and if there is more than one moiety it is a "polylabel".

[0009] In contrast to methods known in the art that focus on making electrospray ionization of analytes more efficient through either mobile phase additives or ESI-enhanced chemical derivatization of the analyte, the present invention increases analyte signal by generating multiple copies of the analyte per analyte molecule. The term "generating a quantifiable signal for an analyte of interest in mass spectrometry" means that a signal is generated in the mass spectrum that corresponds to the analyte of interest but has a higher intensity compared to the intensities of the molecular ion peak of the analyte of interest and its fragmentation peaks, respectively. The quantifiable signal is generated inside the mass spectrometer and originates from the analyte of interest upon fragmentation. Due to the presence of polyunits, the quantifiable signal has either a higher intensity or a higher "fragmentation" efficiency. A "(poly)label" as described herein comprises one or more quantification moieties, all of which are contained within Z, have the same weight (isobaric), and selectively decompose from a precursor molecule into its single repeating components to generate quantification ions. Having multiple copies of isobaric quantification moieties per analyte increases signal intensity and / or fragmentation efficiency in a highly efficient manner. Quantification ions based on individual (poly)labels of formula (I) are shown below as examples: For example, in the case of "Z" of formula (I), which is a nucleoside having adenine as the nucleobase, the quantitative moiety is adenine (elemental composition C5H6N5), which is a 136 Da M-H +1 For example, in the case of "Z" in formula (I), which is a carbamate group, the quantitation moiety is a dehydrated carbamate group (elemental composition C6H13N2O), which produces a 129 Da M H+1 For example, for "Z" in formula (I), which is the tripeptide alanine-proline-glycine (APG), the quantitation moiety is PG (elemental composition C7H12N2O3), which has a molecular weight of 172 Da and a molecular weight of MH. +1 The resulting quantitation ion(s) has the following structure:

[0010] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described herein, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0011] Throughout the text of this specification, several documents are cited. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is incorporated herein by reference in its entirety. In the event of a conflict between a definition or teaching of such an incorporated reference and a definition or teaching cited herein, the text of this specification shall control.

[0012] The elements of the present invention are described below. While these elements are listed with specific embodiments, it is understood that they can be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered to be disclosed by the description of this application unless the context indicates otherwise.

[0013] The word "comprise," and variations such as "comprises" and "comprising," will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.

[0014] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0015] Ratios, concentrations, amounts, and other numerical data may be expressed or presented herein in the form of a "range." It is understood that such range format is used merely for convenience and brevity and, therefore, should be interpreted flexibly to include not only the numerical values ​​explicitly recited as boundaries of the range, but also all individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were explicitly recited. By way of example, a numerical range of "4-20%" should be interpreted not only to include the explicitly recited value 4%-20%, but also to include each individual value and subrange within the stated range. Thus, this numerical range includes individual values ​​such as 4, 5, 6, 7, 8, 9, 10, ..., 18, 19, 20%, etc., and subranges such as 4-10%, 5-15%, 10-20%, etc. This same principle applies to ranges reciting minimum or maximum values. Moreover, such interpretation should apply regardless of the breadth of the range or the described characteristics.

[0016] The term "about," when used in connection with a numerical value, is meant to encompass numerical values ​​within a range having a lower limit of 5% less than the stated numerical value and an upper limit of 5% greater than the stated numerical value.

[0017] The term "mass spectrometry" or "MS" refers to an analytical technique used to identify compounds by their mass. MS is a method of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or "m / z." MS techniques generally involve (1) ionizing compounds to form charged compounds; and (2) detecting the molecular weight of the charged compounds and calculating their mass-to-charge ratio. Compounds can be ionized and detected by any suitable means. A "mass spectrometer" generally includes an ionizer and an ion detector. Generally, one or more molecules of interest are ionized, and the ions are then introduced into a mass spectrometry instrument, where a combination of magnetic and electric fields causes the ions to follow a path in space depending on their mass ("m") and charge ("z"). The term "ionization" or "ionizing" refers to the process of producing ions of an analyte with a net charge equal to one or more electron units. Anions are those that have a net negative charge of one or more electronic units, while cations are those that have a net positive charge of one or more electronic units. MS methods can be performed in either "negative ion mode," in which negative ions are generated and detected, or "positive ion mode," in which positive ions are generated and detected.

[0018] "Tandem mass spectrometry" or "MS / MS" involves multiple steps of selective mass analysis in which analyte fragmentation occurs between steps. In a tandem mass spectrometer, ions are generated in an ion source and separated by mass-to-charge ratio in the first stage of mass analysis (MS1). Ions of specific mass-to-charge ratios (precursor ions or parent ions) are selected, and fragment ions (daughter ions) are generated by collision-induced dissociation, ion-molecule reactions, or photodissociation. The resulting ions are then separated and detected in the second stage of mass analysis (MS2).

[0019] Although ionization sources such as laser desorption ionization (LDI) and atmospheric pressure chemical ionization (APCI) are known, the preferred ionization source in the context of the present invention is electrospray ionization (ESI).

[0020] Mass spectrometers separate and detect ions with slightly different masses, so they can easily distinguish between different isotopes of a given element.Therefore, mass spectrometry is an important method for accurate mass determination and characterization of analytes, including but not limited to low molecular weight analytes, peptides, polypeptides, or proteins.Its applications include the identification of proteins and their post-translational modifications, the elucidation of protein complexes, their subunits, and functional interactions, and the total measurement of proteins in proteomics.De novo sequencing of peptides or proteins by mass spectrometry can usually be performed without prior knowledge of amino acid sequence.

[0021] Mass spectrometric determination may be combined with additional analytical methods, including chromatographic methods such as gas chromatography (GC), liquid chromatography (LC), and high performance liquid chromatography (HPLC), and / or ion mobility-based separation techniques. In the context of this disclosure, the terms "analyte," "analyte molecule," or "analyte of interest" are used interchangeably to refer to chemical species that are analyzed by mass spectrometry. Chemical species, i.e., analytes, suitable for analysis by mass spectrometry can be any type of molecule present in a living organism, including, but not limited to, nucleic acids (e.g., DNA, mRNA, miRNA, rRNA, etc.), amino acids, (poly)peptides, proteins (e.g., cell surface receptors, cytosolic proteins, etc.), metabolites or hormones (e.g., testosterone, estrogen, estradiol, etc.), fatty acids, lipids, carbohydrates, steroids, ketosteroids, secosteroids (e.g., vitamin D), molecules characteristic of specific modifications of other molecules (e.g., sugar moieties or phosphoryl residues on proteins, methyl residues on genomic DNA), or substances internalized by the organism (e.g., therapeutic drugs, drugs of abuse, toxins, etc.), or metabolites of such substances. As also outlined below, preferred analytes of interest are polypeptides and small molecules, more preferably polypeptides. Polypeptides contain 10 or more amino acids coupled to each other via amide bonds. When a polypeptide chain contains more than 100 amino acids and, apart from the primary structure (polypeptide sequence), secondary, tertiary, and sometimes quaternary structures are formed, a polypeptide is called a protein. Preferably, a "polypeptide" in the context of the present invention is a peptide in which in the range of 2 to 100 amino acids are linked by amide bonds.

[0022] With respect to a polypeptide as the analyte of interest, the (poly)label, when bound to the polypeptide, may be attached to the C-terminus, the N-terminus or both termini of the polypeptide.

[0023] In some preferred embodiments of the use of a (poly)label to generate a quantifiable signal for an analyte of interest in mass spectrometry, the (poly)label has the structure of formula (Ia): [ka] During the ceremony, n is an integer selected from the range of 1 to 20; Q is absent or a linker unit; X is a reactive group; Y is [ka] Base, or [ka] a group wherein the dotted line at the oxygen atom indicates the bond to Z and u is either 1 or 2, or [ka] Base, or [ka] Base, or [ka] Base, or [ka] group, wherein the dotted line at the N or C atom in the triazole ring represents the bond to Z, t is 0 or 1; v.1 is 0 or an integer ranging from 1 to 4; v.2 is an integer ranging from 1 to 10; w is 0 or 1; R 1 is a hydrogen atom or a C1-C5 linear or branched alkyl group; R 2 , R3 are independently selected from a hydrogen atom and a C1-C5 straight or branched alkyl group; R 4は , a hydrogen atom or a C1 to C5 linear or branched alkyl group; R 5 is a hydrogen atom; R 6 is absent or is a -C(=O)NH- group or a C(=)O- group or a substituted or unsubstituted C6-C10 arylene, wherein one or more substituents are selected from a hydrogen atom, a halogen atom, and a functional group; R 7 is absent or selected from the group consisting of branched or unbranched C1-C5 alkylene, -O-C1-C5 alkylene, wherein C1-C5 alkylene is branched or unbranched, and -S-C1-C5 alkylene, wherein C1-C5 alkylene is branched or unbranched; Or, R 4 and R 5 together form NR as part of a ring structure 4 form a 5- or 6-membered heteroalkyl ring containing the nitrogen atom; NH or NR for each Y group 4 The dotted line in the figure indicates the Q, the next [YZ] unit, or the Y 1 The dotted line at the C(=O) of each group represents the bond to the next [YZ] unit, Y 1 , or represents a bond to Q; Y 1 teeth, [ka] Base or [ka] a group wherein the dotted line at the oxygen atom indicates the bond to Z and u is either 1 or 2, or [ka] Base, or [ka] basis, or [ka] Base, or [ka] basis, or [ka] Base, or [ka] basis, or [ka] Base, or [ka] It is the basis, In the formula, the dotted line at the N atom or C atom in the triazole ring represents a bond to Z, and t, v.1, v.2, w and R 1 ~R 5 has the same meaning as above for Y; Each Y 1 NH or NR groups 4 or the dotted line at C(=O) represents the bond to the next [YZ] unit; Z is a group consisting of: - a nucleoside having a nucleobase selected from the group consisting of adenine, cytosine, thymine, guanine and uracil; and -Carbamate group -OC(=O)-NH-(CH2) z -N + (Z 3 )3, or -NH-C(=O)-O-(CH2) z -N + (Z 3 ) 3 [wherein z is an integer selected from the range of 1 to 20, Z3 is a C1-C5 alkyl group; and -Tripeptide Z 1 -Proline-Z 2 [In the formula, Z 1 and Z 2 are, independently of one another, amino acids selected from the group consisting of alanine, glycine, valine, leucine, iso-leucine and phenylalanine; Z 2 wherein the C-terminus is preferably blocked, more preferably amidated.

[0024] R 6 When present, R is preferably a substituted or unsubstituted phenylene ring, wherein one or more substituents are selected from hydrogen atoms, halogen atoms and functional groups. 6 is a phenylene group, R 7 or R 7 If there is no -C≡CH, CR 2 R 3 R is bonded to the phenylene at the ortho, meta or para position, preferably the para position, relative to the bond to the adjacent C atom. 6 If CR does not exist, 2 R 3 The adjacent C atom of 7 directly bonded to R 6 and R 7 If both are not present, CR 2 R 3 The adjacent C atom of R is directly bonded to -C≡CH. 6 is a substituted or unsubstituted C6-C10 arylene, R 7 or R 7 When not present, -C≡CH bonds to a free position on the C6-C10 arylene.

[0025] In these embodiments in which the (poly)label has the structure of formula (Ia), the Z groups are not directly attached to the C-terminus and / or N-terminus of the polypeptide, particularly when the analyte of interest is a polypeptide, but rather are always linked to Y, Yi, Y 1and X, and optionally to the Q linker.

[0026] Here and hereinafter, Y or Y containing a triazole ring 1 The structure of is preferably formed by the use of specific amino acids in the synthesis of the (poly)label, which have either an alkyne or azide group in their side chain. Preferably, the precursor of the (poly)label contains an alkyne or azide of the following form (IVa), (IVb): [ka] [ka] During the ceremony, t is 0 or 1; v.1 is 0 or an integer ranging from 1 to 4; v.2 is an integer selected from the range of 1 to 10; R 1 is a hydrogen atom or a C1-C5 linear or branched alkyl group; R 2 , R 3 are independently selected from a hydrogen atom and a C1-C5 straight or branched alkyl group; R 4は , a hydrogen atom or a C1 to C5 linear or branched alkyl group; R 6 is absent or is a -C(=O)NH- group or a C(=)O- group or a substituted or unsubstituted C6-C10 arylene, wherein one or more substituents are selected from a hydrogen atom, a halogen atom, and a functional group; R 7 is absent or selected from the group consisting of branched or unbranched C1-C5 alkylene, -O-C1-C5 alkylene, wherein C1-C5 alkylene is branched or unbranched, and -S-C1-C5 alkylene, wherein C1-C5 alkylene is branched or unbranched; Or, R 4 and R5 together form NR as part of a ring structure 4 Each of (Iva) and (IVb) forms a 5- or 6-membered heteroalkyl ring containing the nitrogen atom of NR 4 or the dotted line at C(=O) represents the bond to the next unit.

[0027] For precursors containing (IVa) or (IVb), R 6 and preferably a substituted or unsubstituted phenylene ring, wherein one or more substituents are selected from hydrogen atoms, halogen atoms and functional groups. 6 is a phenylene group, R 7 or R 7 If there is no -C≡CH, CR 2 R 3 R is bonded to the phenylene at the ortho, meta or para position, preferably the para position, relative to the bond to the adjacent C atom. 6 If CR does not exist, 2 R 3 The adjacent C atom of 7 directly bonded to R 6 and R 7 If both are not present, CR 2 R 3 The adjacent C atom of R is directly bonded to -C≡CH. 6 is a substituted or unsubstituted C6-C10 arylene, R 7 or R 7 When not present, -C≡CH bonds to a free position on the C6-C10 arylene.

[0028] To prepare the (poly)label, the alkyne or azide is then coupled via "click chemistry" with the corresponding azide or alkyne bearing Z to form the (poly)label. In alternative embodiments, the coupling via "click chemistry" occurs before each amino acid bearing an alkyne or azide is coupled to the other via its amino and / or carboxylic acid group, i.e., in these embodiments, structures (IVa) and (IVb) represent separate amino acids, and the NR of each of (Iva), (IVb) 4 The dotted line in represents a bond to a hydrogen atom or an activating group, and the dotted line in C(=O) of each of (Iva) and (IVb) represents a bond to a hydroxyl group or an activating group, respectively.

[0029] "Click chemistry" preferably refers to copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The reaction conditions, catalysts, etc. for such reactions are well known to those skilled in the art.

[0030] In some preferred embodiments of the use of a (poly)label to generate a quantifiable signal for an analyte of interest in mass spectrometry, the (poly)label has the structure of formula (Ia), wherein n is an integer selected from the range of 1 to 20; X is a hydrogen atom; Q is absent; Y is [ka] Base, or [ka] basis, or [ka] Base, or [ka] It is a base; In the formula, NR 4The dotted line in represents a bond to Q or the next [YZ] unit, respectively, and the dotted line in C(=O) represents a bond to the next [YZ] unit or Y 1 The dotted line at the N atom or C atom in the triazole ring represents a bond to Z, and t, v.1, v.2, w and R 1 ~R 5 has the same meaning as given above in embodiment 2; Y 1 teeth, [ka] Base, or [ka] basis, or [ka] Base, or [ka] basis, or [ka] Base, or [ka] basis, or [ka] Base, or [ka] It is a base; In the formula, NR 4 or C(=O) represents a bond to the next [YZ] unit, and dotted lines at N or C atoms in the triazole ring represent bonds to Z, and t, v.1, v.2, w, and R 1 ~R 5has the same meaning as given above in embodiment 2; and Z is a nucleoside having a nucleobase selected from the group consisting of adenine, cytosine, thymine, guanine and uracil.

[0031] In some preferred embodiments of the use of a (poly)label to generate a quantifiable signal for an analyte of interest in mass spectrometry, the (poly)label has the structure of formula (Ia), where n is an integer selected from the range of 1 to 20; Q is a linker unit, and X is a reactive group; Y is [ka] Base, or [ka] basis, or [ka] Base, or [ka] It is the basis, In the formula, NR 4 The dotted line in represents a bond to Q or the next [YZ] unit, respectively, and the dotted line in C(=O) represents a bond to the next [YZ] unit or Y 1 The dotted line at the N atom or C atom in the triazole ring represents a bond to Z, and t, v.1, v.2, w and R 1 ~R 5 has the same meaning as given above in embodiment 2; Y 1 teeth, [ka] Base, or [ka] basis, or [ka] Base, or [ka] basis, or [ka] Base, or [ka] basis, or [ka] Base, or [ka] It is a base; In the formula, NR 4 or C(=O) represents a bond to the next [YZ] unit, and dotted lines at N or C atoms in the triazole ring represent bonds to Z, and t, v.1, v.2, w, and R 1 ~R 5 has the same meaning as given above in embodiment 2; and Z is a carbamate group -OC(=O)-NH-(CH2) z -N + (Z 3 )3 or -NH-C(=O)-O-(CH2) z -N + (Z 3 ) 3, wherein z is an integer selected from the range of 1 to 10, and Z 3 is a C1 to C5 alkyl group.

[0032] In some preferred embodiments of the use of (poly)labels to generate a quantifiable signal for an analyte of interest in mass spectrometry, The (poly)label has a structure of formula (Ia), where n is an integer selected from the range of 1 to 20; Q is absent or is a linker unit; X is a hydrogen atom or a reactive group; Y is [ka] group, where the dotted line at the oxygen atom indicates a bond to Z, u is 1 or 2, the dotted line at NH represents a bond to Q or the next [YZ] unit, respectively, and the dotted line at C(═O) represents a bond to the next [YZ] unit or Y, respectively. 1 represents a bond to; Y 1 teeth, [ka] Base, or [ka] It is the basis, wherein the dotted line at the oxygen atom represents the bond to Z, u is 1 or 2, and the dotted line at the NH or C(=O) represents the bond to the next [YZ] unit; and Z is tripeptide Z 1 -Proline-Z 2 [In the formula, Z 1 and Z 2 are, independently of one another, amino acids selected from the group consisting of alanine, glycine, valine, leucine, iso-leucine and phenylalanine; Z 2 The C-terminus of is preferably blocked, more preferably amidated.

[0033] In some preferred embodiments of the use of (poly)labels to generate a quantifiable signal for an analyte of interest in mass spectrometry, Y is [ka] It is the basis, where the dotted line at the oxygen atom indicates a bond to Z, u is either 1 or 2, the dotted line at NH represents a bond to Q or the next [YZ] unit, respectively, and the dotted line at C(=O) represents a bond to the next [YZ] unit or Y, respectively. 1 represents a bond to, or [ka] Base, or [ka] It is the basis, In the formula, the dotted line at the N atom or C atom in the triazole ring represents a bond to Z, and R 1 is a hydrogen atom or a methyl group, and the dotted line at the NH of each group indicates a link to Q, to the next [YZ] unit, or to Y 1 The dotted line at the C(=O) of each group represents the bond to the next [YZ] unit, Y 1 or to Q, and u is 1 or 2.

[0034] In some preferred embodiments of the use of (poly)labels to generate a quantifiable signal for an analyte of interest in mass spectrometry, Y 1 teeth, [ka] Base, or [ka] It is the basis, wherein the dotted line at the oxygen atom represents the bond to Z, u is 1 or 2, and the dotted line at the NH or C(=O) represents the bond to the next [YZ] unit, or [ka] Base, or [ka] basis, or [ka] Base, or [ka] basis, or [ka] ,or [ka] basis, or [ka] Base, or [ka] It is the basis, In the formula, the dotted line at the NH or C(=O) represents a bond to the next [YZ] unit, the dotted line at the N atom or C atom in the triazole ring represents a bond to Z, and R 1 is a methyl group.

[0035] In some preferred embodiments of the use of a (poly)label to generate a quantifiable signal for an analyte of interest in mass spectrometry, in the (poly)label structure of formula (I), n and m are both 0, Q is absent, and X is a hydrogen atom, and the (poly)label has the structure of formula (Ib): Z 1 -Proline-Z 2 (Ib) In the formula, Z 1 and Z 2 are, independently of one another, amino acids selected from the group consisting of alanine, glycine, valine, leucine, iso-leucine and phenylalanine; Z 2 The C-terminus of is preferably blocked, more preferably amidated.

[0036] In some preferred embodiments of the use of (poly)labels to generate a quantifiable signal for an analyte of interest in mass spectrometry, Z 1 and Z 2 are independently selected from alanine and glycine, preferably Z 1 is alanine and Z 2 is glycine.

[0037] In those embodiments in which the (poly)label has the structure of formula (Ib), the Z group is preferably attached directly to the C-terminus and / or N-terminus of the polypeptide, particularly when the analyte of interest is a polypeptide.

[0038] In some preferred embodiments of the use of (poly)labels to generate a quantifiable signal for an analyte of interest in mass spectrometry, n is an integer selected from the range of 1 to 10, preferably from the range of 2 to 8.

[0039] In some preferred embodiments of the use of (poly)labels to generate a quantifiable signal for an analyte of interest in mass spectrometry, R of each Y of the n[YZ] units 1 and Y 1 R 1 are each hydrogen atoms.

[0040] In some preferred embodiments of the use of (poly)labels to generate a quantifiable signal for an analyte of interest in mass spectrometry, R of each Y of the n[YZ] units 1 and Y 1 R 1 are each a methyl group.

[0041] In some preferred embodiments of the use of a (poly)label to generate a quantifiable signal for an analyte of interest in mass spectrometry, when Z is a nucleoside, the nucleoside has the structure (IIa), (IIb), or (IIc): [ka] [ka] [ka] In the formula, the dotted line at position 1 of the five-membered ring represents a bond to a nucleobase, and the dotted lines at the CH2 units at position 3 of the five-membered ring or position 4 of the five-membered ring represent Y and Y, respectively. 1 and preferably Y and Y respectively. 1 represents a bond to the N atom in the triazole ring; R, R' are independently a hydrogen atom or a -CH2-P(=O)(OH)2 group.

[0042] In some preferred embodiments of the use of (poly)labels to generate a quantifiable signal for an analyte of interest in mass spectrometry, the nucleobase of nucleoside Z is adenine.

[0043] In some preferred embodiments of the use of (poly)labels to generate a quantifiable signal for an analyte of interest in mass spectrometry, Z is a carbamate group -OC(=O)-NH-(CH) z -N + (CH3)3 or -NH-C(=O)-O-(CH2) z -N + (Z 3 )3, in which z is an integer selected from the range of 1 to 10, preferably selected from the range of 2 to 5, and more preferably z is 2.

[0044] In some preferred embodiments of the use of (poly)labels to generate a quantifiable signal for an analyte of interest in mass spectrometry, Q is (C1-C5 alkylene-O-) runits [r is an integer in the range of 1 to 10] or C1-C20 alkanediyl units or C1-C20 alkanediyl-heteroaryl units or (C1-C5 alkanediyl)-O-(C1-C5 alkanediyl) units or C2-C9 alkanediyl-C(=O) units or C2-C9 alkanediyl-NH units, preferably C4-C7 alkanediyl-C(=O) units or C4-C7 alkanediyl-NH units, more preferably hexylene-C(=O) units or hexylene-NH units, and [NH-C1-C5 alkanediyl-C(=O)] x unit or [C(=O)-C1-C5 alkanediyl-NH] x A linker comprising the unit [wherein x is an integer selected from the range of 1 to 20, preferably the range of 2 to 8, more preferably the range of 3 to 7, more preferably the range of 4 to 6; and / or C1-C5 alkanediyl is preferably C2-C4 alkanediyl, more preferably ethylene].

[0045] In some preferred embodiments of the use of (poly)labels to generate a quantifiable signal for an analyte of interest in mass spectrometry, X is a reactive group selected from the group consisting of an isothiocyanate group, an isocyanate group, an acyl azide group, a sulfonyl chloride group, an aldehyde group, a glyoxal group, an epoxide group, an oxirane group, a carbonate group, an aryl halide group, an imidoester group, a carbodiimide group, an anhydride group, a fluorophenyl ester group, a carboxyl group, a HATU ester group, a HBTU ester group and an NHS ester group, preferably an NHS ester group.

[0046] Second Aspect—Process for Producing a Quantifiable Signal for an Analyte of Interest In a second aspect, the present invention relates to a process for modifying an analyte of interest to obtain an increased intensity signal in mass spectrometry, comprising the steps of: (a) providing at least one (poly)label having a reactive group of structure (I), [ka] During the ceremony, m is 0 or 1; n is 0 or an integer selected from the range of 1 to 20; Q is absent or a linker unit; X is a reactive group or, if Q is absent, a hydrogen atom; Y is an amino acid-based linker unit having a side chain suitable for coupling to Z; Y 1 is a linker unit based on an amino acid having a side chain suitable for coupling to Z, the amino acid having a blocked carboxylic acid group or a blocked amino group; Z is a group consisting of: - a nucleoside comprising a nucleobase selected from the group consisting of: adenine, cytosine, thymine, guanine and uracil; -Structure Z 1 -Proline-Z 2 A tripeptide of the formula: 1 and Z 2 are, independently of each other, amino acids selected from the group consisting of alanine, glycine, valine, leucine, iso-leucine and phenylalanine; and -Carbamate group -OC(=O)-NH-(CH2) z -N + (Z 3 )3 or -NH-C(=O)-O-(CH2) z -N + (Z 3 ) 3 [wherein z is an integer selected from the range of 1 to 10, Z 3 is a C1-C5 alkyl group; (b) providing an analyte of interest, the analyte being selected from the group consisting of polypeptides and small molecules, preferably a polypeptide having free amino groups and / or free carboxyl groups, which, if present, are optionally activated; (c) reacting at least one (poly)label having a reactive group with the analyte of interest, thereby obtaining a reaction product, wherein the at least one (poly)label is covalently bound to the analyte of interest.

[0047] All details, embodiments and preferred embodiments described above in the section relating to the first aspect also apply to the second aspect of the invention.

[0048] In some preferred embodiments of the process for modifying an analyte of interest to obtain an increased intensity signal in mass spectrometry, the analyte of interest is a small molecule that is an organic compound having a molecular weight of ≦1000 Daltons, and the small molecule is preferably a drug.

[0049] In some preferred embodiments of the process for modifying an analyte of interest to obtain an increased intensity signal in mass spectrometry, the analyte of interest is a polypeptide having free amino groups and / or free carboxyl groups, and the reaction product obtained in (c) is a compound having the general structural formula (III): [ka] In the formula, Q, Y, Y 1 , Z, m and n have the meanings defined above in relation to the first aspect of the invention, and X a , X b are each the remainder of a group X as defined above in relation to the first aspect of the invention after forming a bond, preferably a covalent bond, with the corresponding functional group of the polypeptide, and y and y are each 0 or 1, with the proviso that at least one of x, y is 1; and R is the remainder of the polypeptide.

[0050] As indicated above in the section relating to the first aspect, a "polypeptide" is a peptide in which 2 to 100 amino acids are linked by amide bonds. When R is a residue of a polypeptide, the "residue" means that the C-terminal carboxyl group and / or N-terminal amino group of the peptide form an amide bond with the reactive group of the (poly)label. When x is 0, the N-terminus of the polypeptide is still an -NH2 group. When x is 1, there is a residue of an -NH2 group, such as -NH-, at the N-terminus of the polypeptide. When y is 0, there is still a COOH group at the C-terminus of the polypeptide. When y is 1, there is a residue of a COOH group, such as -C(=O)-, at the C-terminus of the polypeptide.

[0051] Third Aspect—Method of Determining an Analyte of Interest by Mass Spectrometry A third aspect of the present invention relates to a method for determining an analyte of interest by mass spectrometry, comprising: (i) providing a reaction product of the analyte of interest, the reaction product being based on a (poly)label having a structural element (I), [ka] covalently attached to the analyte of interest, wherein Q, X, Y, Y 1 , Y 2 wherein Z, m and n have the meanings defined in the sections relating to the first and second aspects of the invention above; (ii) subjecting the reaction product provided in (i) to mass spectrometry; (iii) (unsubstituted) Z-MH +1 determining the intensity in the mass spectrum of the fragment corresponding to the peak or its water depletion product.

[0052] All details, embodiments and preferred embodiments described above in the sections relating to the first and second aspects also apply to the third aspect of the invention.

[0053] Fourth embodiment - Polylabeling A fourth aspect of the present invention relates to a (poly)label having the structure of formula (I): [ka] In the formula, Q, X, Y, Y 1 , Y 2 , Z, n and m have the meanings defined in the section relating to the first aspect of the invention above.

[0054] All details, embodiments and preferred embodiments discussed above in the sections relating to the first, second and third aspects also apply to the fourth aspect of the invention.

[0055] Fifth Aspect—Reaction Products In a fifth aspect, the present invention relates to a reaction product comprising a polypeptide and a (poly)label having the general structural formula (III): [ka] In the formula, Q, X, Y, Y 1 , Y 2 , Z, n and m have the meanings defined in the sections relating to the first, second, third and / or fourth aspects above, and the designations x, y are either 0 or 1, provided that at least one of x, y is 1, and R is the remainder of the polypeptide.

[0056] All details, embodiments and preferred embodiments discussed above in the sections relating to the first, second, third and fourth aspects also apply to the fifth aspect of the invention.

[0057] The present invention is further described by the following embodiments and combinations of embodiments, as indicated by their respective dependencies and back references. In particular, in each example where various embodiments are listed, in relation to a term such as "described in any one of embodiments 1 to 4," it is meant that all embodiments within this scope are clearly disclosed to one skilled in the art, i.e., this expression is understood by one skilled in the art to be synonymous with "described in any one of embodiments 1, 2, 3, and 4."

[0058] 1. The use of a (poly)label to generate a quantifiable signal for an analyte of interest in mass spectrometry, wherein the (poly)label has the structure (I): [ka] During the ceremony, m is 0 or 1; n is 0 or an integer selected from the range of 1 to 20; Q is absent or a linker unit; X is a reactive group or, if Q is absent, a hydrogen atom; Y is an amino acid-based linker unit having a side chain suitable for coupling to Z; Y 1 is a linker unit based on an amino acid having a side chain suitable for coupling to Z, the amino acid having a blocked carboxylic acid group or a blocked amino group; Z is a group consisting of: - a nucleoside comprising a nucleobase selected from the group consisting of: adenine, cytosine, thymine, guanine and uracil; -Structure Z 1 -Proline-Z 2 A tripeptide of the formula: 1 and Z 2 are, independently of each other, amino acids selected from the group consisting of alanine, glycine, valine, leucine, iso-leucine and phenylalanine; and -Carbamate group -OC(=O)-NH-(CH2) z -N + (Z 3 )3 or -NH-C(=O)-O-(CH2) z -N + (Z 3 ) 3 [wherein z is an integer selected from the range of 1 to 10, Z 3 is a C1-C5 alkyl group.

[0059] 2. The use according to embodiment 1, wherein the (poly)label has the structure of formula (Ia): [ka] During the ceremony, n is an integer selected from the range of 1 to 20; Q is absent or a linker unit; X is a reactive group; Y is [ka] Base, or [ka] a group wherein the dotted line at the oxygen atom indicates the bond to Z and u is either 1 or 2, or [ka] Base, or [ka] basis, or [ka] Base, or [ka] group, wherein the dotted line at the N or C atom in the triazole ring represents the bond to Z, t is 0 or 1; v.1 is 0 or an integer ranging from 1 to 4; v.2 is an integer ranging from 1 to 10; w is 0 or 1; R 1 is a hydrogen atom or a C1-C5 linear or branched alkyl group; R 2 , R 3 are independently selected from a hydrogen atom and a C1-C5 straight or branched alkyl group; R 4は , a hydrogen atom or a C1 to C5 linear or branched alkyl group; R 5 is a hydrogen atom; R 6 is absent or is a -C(=O)NH- group or a C(=)O- group or a substituted or unsubstituted C6-C10 arylene, wherein one or more substituents are selected from a hydrogen atom, a halogen atom, and a functional group; R 7 is absent or selected from the group consisting of branched or unbranched C1-C5 alkylene, -O-C1-C5 alkylene, wherein C1-C5 alkylene is branched or unbranched, and -S-C1-C5 alkylene, wherein C1-C5 alkylene is branched or unbranched; Or, R 4 and R 5 together form NR as part of a ring structure 4 form a 5- or 6-membered heteroalkyl ring containing the nitrogen atom; NH or NR for each Y group 4 The dotted line in the figure indicates the Q, the next [YZ] unit, or the Y 1 The dotted line at the C(=O) of each group represents the bond to the next [YZ] unit, Y 1 , or represents a bond to Q; Y 1 teeth, [ka] Base or [ka] a group wherein the dotted line at the oxygen atom indicates the bond to Z and u is either 1 or 2, or [ka] Base, or [ka] basis, or [ka] Base, or [ka] basis, or [ka] Base, or [ka] basis, or [ka] Base, or [ka] It is the basis, In the formula, the dotted line at the N atom or C atom in the triazole ring represents a bond to Z, and t, v.1, v.2, w and R 1 ~R 5 has the same meaning as above for Y; Each Y 1 NH or NR groups 4 or the dotted line at C(=O) represents the bond to the next [YZ] unit; Z is a group consisting of: - a nucleoside having a nucleobase selected from the group consisting of adenine, cytosine, thymine, guanine and uracil; and -Carbamate group -OC(=O)-NH-(CH2) z -N + (Z 3 )3, or -NH-C(=O)-O-(CH2) z -N + (Z 3 ) 3 [wherein z is an integer selected from the range of 1 to 20, Z 3 is a C1-C5 alkyl group; and -Tripeptide Z 1 -Proline-Z 2 [In the formula, Z 1 and Z 2 are, independently of one another, amino acids selected from the group consisting of alanine, glycine, valine, leucine, iso-leucine and phenylalanine; Z 2 wherein the C-terminus is preferably blocked, more preferably amidated.

[0060] 3. The use according to embodiment 1 or 2, wherein the (poly)label has a structure of formula (Ia), where n is an integer selected from the range of 1 to 20; X is a hydrogen atom; Q is absent; Y is [ka] Base, or [ka] basis, or [ka] Base, or [ka] It is a base; In the formula, NR 4The dotted line in represents a bond to Q or the next [YZ] unit, respectively, and the dotted line in C(=O) represents a bond to the next [YZ] unit or Y 1 The dotted line at the N atom or C atom in the triazole ring represents a bond to Z, and t, v.1, v.2, w and R 1 ~R 5 has the same meaning as given above in embodiment 2; Y 1 teeth, [ka] Base, or [ka] basis, or [ka] Base, or [ka] basis, or [ka] Base, or [ka] basis, or [ka] Base, or [ka] It is a base; In the formula, NR 4 or C(=O) represents a bond to the next [YZ] unit, and dotted lines at N or C atoms in the triazole ring represent bonds to Z, and t, v.1, v.2, w, and R 1 ~R 5has the same meaning as given above in embodiment 2; and Z is a nucleoside having a nucleobase selected from the group consisting of adenine, cytosine, thymine, guanine and uracil.

[0061] 4. The use according to embodiment 1 or 2, wherein the (poly)label has a structure of formula (Ia), where n is an integer selected from the range of 1 to 20; Q is a linker unit, and X is a reactive group; Y is [ka] Base, or [ka] basis, or [ka] Base, or [ka] It is the basis, In the formula, NR 4 The dotted line in represents a bond to Q or the next [YZ] unit, respectively, and the dotted line in C(=O) represents a bond to the next [YZ] unit or Y 1 The dotted line at the N atom or C atom in the triazole ring represents a bond to Z, and t, v.1, v.2, w and R 1 ~R 5 has the same meaning as given above in embodiment 2; Y 1 teeth, [ka] Base, or [ka] basis, or [ka] Base, or [ka] basis, or [ka] Base, or [ka] Base, or [ka] Base, or [ka] It is a base; In the formula, NR 4 or C(=O) represents a bond to the next [YZ] unit, and dotted lines at N or C atoms in the triazole ring represent bonds to Z, and t, v.1, v.2, w, and R 1 ~R 5 has the same meaning as given above in embodiment 2; and Z is a carbamate group -OC(=O)-NH-(CH2) z -N + (Z 3 )3 or -NH-C(=O)-O-(CH2) z -N + (Z 3 ) 3, wherein z is an integer selected from the range of 1 to 10, and Z 3 is a C1 to C5 alkyl group.

[0062] 5. The use according to embodiment 1 or 2, wherein the (poly)label has a structure of formula (Ia), where n is an integer selected from the range of 1 to 20; Q is absent or is a linker unit; X is a hydrogen atom or a reactive group; Y is [ka] group, where the dotted line at the oxygen atom indicates a bond to Z, u is 1 or 2, the dotted line at NH represents a bond to Q or the next [YZ] unit, respectively, and the dotted line at C(═O) represents a bond to the next [YZ] unit or Y, respectively. 1 represents a bond to; Y 1 teeth, [ka] Base, or [ka] It is the basis, wherein the dotted line at the oxygen atom represents the bond to Z, u is 1 or 2, and the dotted line at the NH or C(=O) represents the bond to the next [YZ] unit; and Z is tripeptide Z 1 -Proline-Z 2 [In the formula, Z 1 and Z 2 are, independently of one another, amino acids selected from the group consisting of alanine, glycine, valine, leucine, iso-leucine and phenylalanine; Z 2 The C-terminus of is preferably blocked, more preferably amidated.

[0063] 6. The use according to any one of embodiments 2 to 5, Y is [ka] It is the basis, where the dotted line at the oxygen atom indicates a bond to Z, u is either 1 or 2, the dotted line at NH represents a bond to Q or the next [YZ] unit, respectively, and the dotted line at C(=O) represents a bond to the next [YZ] unit or Y, respectively. 1 represents a bond to, or [ka] Base, or [ka] It is the basis, In the formula, the dotted line at the N atom or C atom in the triazole ring represents a bond to Z, and R 1 is a hydrogen atom or a methyl group, and the dotted line at the NH of each group indicates a link to Q, to the next [YZ] unit, or to Y 1 The dotted line at the C(=O) of each group represents the bond to the next [YZ] unit, Y 1 or to Q, and u is 1 or 2.

[0064] 7. The use according to any one of embodiments 2 to 6, Y 1 teeth, [ka] Base, or [ka] It is the basis, wherein the dotted line at the oxygen atom represents the bond to Z, u is 1 or 2, and the dotted line at the NH or C(=O) represents the bond to the next [YZ] unit, or [ka] Base, or [ka] basis, or [ka] Base, or [ka] basis, or [ka] ,or [ka] basis, or [ka] Base, or [ka] It is the basis, In the formula, the dotted line at the NH or C(=O) represents a bond to the next [YZ] unit, the dotted line at the N atom or C atom in the triazole ring represents a bond to Z, and R 1 is a methyl group.

[0065] 8. The use according to embodiment 1 or 2, wherein in the (poly)label structure of formula (I), n and m are both 0, Q is absent, and X is a hydrogen atom, and the (poly)label has the structure of formula (Ib): Z 1 -Proline-Z 2 (Ib) In the formula, Z 1 and Z 2 are, independently of one another, amino acids selected from the group consisting of alanine, glycine, valine, leucine, iso-leucine and phenylalanine; Z 2 The C-terminus of is preferably blocked, more preferably amidated.

[0066] 9.Z 1 and Z 2 are independently selected from alanine and glycine, preferably Z 1 is alanine and Z 2 The use according to any one of embodiments 1 to 8, wherein is glycine.

[0067] 10. Use according to any one of embodiments 1 to 9, wherein n is an integer selected from the range of 1 to 10, preferably from the range of 2 to 8.

[0068] 11. R of each Y in n [YZ] units 1 and Y 1 R 1 The use of any one of embodiments 1 to 10, wherein each is a carbon atom.

[0069] 12. R of each Y in n [YZ] units 1 and Y 1 R 1 The use of any one of embodiments 1 to 9, wherein each is a methyl group.

[0070] 13. The use according to any one of embodiments 1 to 12, wherein Z is a nucleoside, the nucleoside has the structure (IIa), (IIb), or (IIc): [ka] [ka] [ka] In the formula, the dotted line at position 1 of the five-membered ring represents a bond to a nucleobase, and the dotted lines at the CH2 units at position 3 of the five-membered ring or position 4 of the five-membered ring represent Y and Y, respectively. 1 and preferably Y and Y respectively. 1 represents the bond to the N atom in the triazole ring; R, R' are independently a hydrogen atom or a -CH2-P(=O)(OH)2 group, as used.

[0071] 14. The use according to any one of embodiments 1 to 13, wherein the nucleobase of nucleoside Z is adenine.

[0072] 15.Z is a carbamate group -OC(=O)-NH-(CH2) z -N + (CH3)3 or -NH-C(=O)-O-(CH2) z -N + (Z 3)3, wherein z is an integer selected from the range of 1 to 10, preferably selected from the range of 2 to 5, more preferably z is 2.

[0073] 16. The use according to any one of embodiments 1 to 15, wherein Q is (C1-C5 alkylene-O-) r units [r is an integer ranging from 1 to 10] or C1-C20 alkanediyl units or C1-C20 alkanediyl-heteroaryl units or (C1-C5 alkanediyl)-O-(C1-C5 alkanediyl) units or C2-C9 alkanediyl-C(=O) units or C2-C9 alkanediyl-NH units, preferably C4-C7 alkanediyl-C(=O) units or C4-C7 alkanediyl-NH units, more preferably hexylene-C(=O) units or hexylene-NH units, and [NH-C1-C5 alkanediyl-C(=O)] x unit or [C(=O)-C1-C5 alkanediyl-NH] x The use of a linker comprising a unit [wherein x is an integer selected from the range of 1 to 20, preferably the range of 2 to 8, more preferably the range of 3 to 7, more preferably the range of 4 to 6; and / or the C1-C5 alkanediyl is preferably a C2-C4 alkanediyl, more preferably ethylene].

[0074] 17. Use according to any one of embodiments 1 to 16, wherein X is a reactive group selected from the group consisting of an isothiocyanate group, an isocyanate group, an acyl azide group, a sulfonyl chloride group, an aldehyde group, a glyoxal group, an epoxide group, an oxirane group, a carbonate group, an aryl halide group, an imide ester group, a carbodiimide group, an anhydride group, a fluorophenyl ester group, a carboxyl group, a HATU ester group, a HBTU ester group and an NHS ester group, preferably an NHS ester group.

[0075] 18. A process for modifying an analyte of interest to obtain an increased intensity signal in mass spectrometry, comprising the steps of: (b) (a) providing at least one (poly)label having a reactive group of structure (I), [ka] During the ceremony, m is 0 or 1; n is 0 or an integer selected from the range of 1 to 20; Q is absent or a linker unit; X is a reactive group or, if Q is absent, a hydrogen atom; Y is an amino acid-based linker unit having a side chain suitable for coupling to Z; Y 1 is a linker unit based on an amino acid having a side chain suitable for coupling to Z, the amino acid having a blocked carboxylic acid group or a blocked amino group; Z is a group consisting of: - a nucleoside comprising a nucleobase selected from the group consisting of: adenine, cytosine, thymine, guanine and uracil; -Structure Z 1 -Proline-Z 2 A tripeptide of the formula: 1 and Z 2 are, independently of each other, amino acids selected from the group consisting of alanine, glycine, valine, leucine, iso-leucine and phenylalanine; and -Carbamate group -OC(=O)-NH-(CH2) z -N + (Z 3 )3 or -NH-C(=O)-O-(CH2) z -N + (Z 3 ) 3 [wherein z is an integer selected from the range of 1 to 10, Z 3 is a C1-C5 alkyl group; (b) providing an analyte of interest, the analyte being selected from the group consisting of polypeptides and small molecules, preferably a polypeptide having free amino groups and / or free carboxyl groups, which, if present, are optionally activated; (c) reacting at least one (poly)label having a reactive group with an analyte of interest, thereby obtaining a reaction product, wherein the at least one (poly)label is covalently bound to the analyte of interest.

[0076] 19. The process of embodiment 18, wherein the analyte of interest is a small molecule that is an organic compound having a molecular weight of ≦1000 Daltons, and the small molecule is preferably a drug.

[0077] 20. The process according to embodiment 18, wherein the analyte of interest is a polypeptide having a free amino group and / or a free carboxyl group, and the reaction product obtained in (c) is a compound having the general structural formula (III): [ka] In the formula, Q, Y, Y 1 , Z, m and n have the meanings defined in any one of embodiments 1 to 17, and X a , X b are each the remainder of group X as defined in any one of embodiments 1 to 17 after forming a bond, preferably a covalent bond, with the corresponding functional group of the polypeptide; y and y are each 0 or 1, with the proviso that at least one of x, y is 1; and R is the remainder of the polypeptide.

[0078] 21. A method for determining an analyte of interest by mass spectrometry, comprising: (i) providing a reaction product of the analyte of interest, the reaction product being based on a (poly)label having a structural element (I), [ka] During the ceremony, covalently attached to the analyte of interest, wherein Q, X, Y, Y 1 , Y 2 wherein Z, m and n have the meanings defined in any one of the above embodiments; (ii) subjecting the reaction product provided in (i) to mass spectrometry; (iii) (unsubstituted) Z-MH +1 determining the intensity in the mass spectrum of the fragment corresponding to the peak or its water depletion product.

[0079] 22. A (poly)label having the structure of formula (I), [ka] In the formula, Q, X, Y, Y 1 , Y 2 , Z, n and m have the meanings defined in any one of the above embodiments.

[0080] 23. A reaction product comprising a polypeptide and a (poly)tag having the general structural formula (III), [ka] In the formula, Q, X, Y, Y 1 , Y 2 , Z, n, and m have the meaning defined in any one of the above embodiments, and the designations x, y are either 0 or 1, provided that at least one of x, y is 1, and R is the remainder of a polypeptide. [Brief explanation of the drawings]

[0081] [Figure 1] The MS / MS spectrum of the model peptide (ATNSQLFR) is shown. The letters above the peaks indicate peptide fragments according to the peptide fragmentation nomenclature. [Figure 2]The MS / MS spectrum of the synthetic peptide (poly)labeled construct (ATNSQLFR-P) is shown. "P" indicates an (adenine-based) (poly)labeled construct. The "quantitative ion" here refers to adenine (C5H6N5) with MH+1 (Da) = 136. [Figure 3] The MS / MS spectrum of the synthetic peptide (poly)labeled construct (ATNSQLFR-P) is shown. "P" indicates an APG-based (poly)labeled construct. ATNSQLFR~P* indicates a peptide-(poly)labeled construct lacking the moiety, where the "moiety" was PG (proline-glycine, C7H12N2O3, MH+1 (Da) = 172). [Figure 4] The MS / MS spectrum of the synthetic peptide (poly)labeled construct (ATNSQLFR-P) is shown. "P" indicates a carbamate-based (poly)labeled construct. ATNSQLFR~P* indicates a peptide-(poly)labeled construct lacking a quantitative ion, where "moiety" is O=CNH-CH2-CH-N+(CH3)3, and MH+1 (Da) = 129. [Figure 5] Figure 1 shows MS / MS spectra of peptide (poly)labeled constructs containing (poly)tags at the C- and N-termini of the constructs. "P~ATNSQLFR~P" indicated an intact peptide (poly)label with two quantification ions. P*~ATNSQLFR~P indicated a peptide (poly)label with one quantification ion removed. P*~ATNSQLFR~P* indicated a peptide (poly)labeled construct with two quantification ions removed. Detection of the peptide (poly)labeled construct (P*~ATNSQLFR~P*) demonstrated that multiple fragmentation events occurred for individual peptide analytes. "Quantification ion" here refers to adenine (C5H6N5) with MH+1 (Da) = 136. [Figure 6]MS / MS spectra of peptide (poly)labeled constructs containing APG-based (poly)tags at the C and N termini of the constructs are shown. P*~ATNSQLFR~P indicated a peptide (poly)labeled construct with one quantification ion removed. P*~ATNSQLFR~P* indicated a peptide (poly)labeled construct with two quantification ions removed. Detection of the peptide (poly)labeled construct (P*~ATNSQLFR~P*) demonstrated that multiple fragmentation events occurred in individual peptide analytes, and the "moiety" was PG (proline-glycine, C7H12N2O3, MH+1 (Da) = 172). [Figure 7] The MS / MS spectrum of the carbamate-based peptide-2x((poly)labeled) construct is shown. Fragmentation was optimized to detect the intact ATNSQLFR~P~P, fragment ATNSQLFR~P~P*, and ATNSQLFR~P*~P* ions. ATNSQLFR~P~P indicates the intact peptide-2x((poly)labeled) construct, ATNSQLFR~P~P* indicates the remaining fragment ion with the 147 Da fragment missing, and ATNSQLFR~P*~P* indicates the remaining fragment ion lacking the 2x147 Da fragment, where "part" is O=CNH-CH2-CH-N+(CH3)3, and MH+1 (Da) = 129. [Figure 8] Relative SRM signal intensities of unmodified synthetic peptides as 1x and 2x (poly)labeled constructs for adenine-based (poly)labeling are shown. SRM intensities were normalized to the SRM intensity of the unmodified peptide. The SRM intensity of the unmodified peptide was monitored at the most abundant fragment ion (y6 ion, 764.6 m / z), and the (poly)labeled peptide was monitored at the "quantitation ion" with M−H+1(Da)=136 (adenine, C5H6N5). [Figure 9]Relative SRM signal intensities of unmodified synthetic peptides as 1x and 2x (poly)labeled constructs for APG-based (poly)labeling are shown. SRM intensities were normalized to the SRM intensity of the unmodified peptide. The SRM intensity of the unmodified peptide was monitored at the most abundant fragment ion (y6 ion, 764.6 m / z), and the (poly)labeled peptide was monitored at the "quantitation ion" with M−H+1 (Da)=172 (PG, lysine-glycine, C7H12N2O3). [Figure 10] The relative fragmentation efficiencies of unmodified synthetic peptides as 1x and 2x (poly)labeled constructs for adenine-based (poly)labeling are shown. Individual fragmentation efficiencies were normalized to that of the unmodified peptide. [Figure 11] Figure 1 shows the relative fragmentation efficiencies of unmodified synthetic peptides as 1x and 2x (poly)labeled constructs for APG-based (poly)labeling. Individual fragmentation efficiencies were normalized to that of the unmodified peptide. [Figure 12] Relative SRM signal intensities of synthetic unmodified carbamate-based 1x-, 2x-, 4x-, and 6x-(poly)label-containing peptide constructs are shown. SRM intensities were normalized to that of the unmodified peptide. The unmodified peptide was monitored at its most abundant fragment ion (y6 ion, m / z 764.6), and the (poly)labeled peptide was monitored at the "quantitation ion" with MH+1 (Da) = 129 (O = CNH-CH2-CH-N+(CH3)3). [Figure 13] The relative fragmentation efficiencies of carbamate-based 1x-, 2x-, 4x-, and 6x-(poly)label-containing peptide constructs are shown: individual fragmentation efficiencies were normalized to that of the unmodified peptide. [Figure 14]Figure 1 shows the relative fragmentation efficiencies of derivatized tryptic peptides with NHS-ester-containing carbamate-based 2x and 4x-((poly)-labeled) peptides. The fragmentation efficiency of each individual peptide-(poly)-labeled construct was normalized to that of peptide-2x((poly)-labeled).

[0082] The present invention will be further illustrated by the following Reference Examples, Comparative Examples and Examples. [Example]

[0083] Reference Example 1: Dissolution of synthetic peptides Gravimetrically determined synthetic peptides were dissolved at a concentration of 1 μM using 0.1% CH 2 O 2 , 5% C 2 H 3 N in H 2 O.

[0084] Reference Example 2: Liquid chromatography tandem mass spectrometry (LC-MS / MS) Adjusting MS parameters A triple quadrupole mass spectrometer was tuned for each synthetic (poly)labeled peptide by using a direct injection strategy, combining the flows from syringe pumps using a T-junction to deliver a 1 μM (poly)labeled peptide solution at a flow rate of 5 μL / min and the LC flow (295 μL / min).

[0085] HPLC parameters Vanquish UHPLC Autosampler, Thermo Scientific Vanquish UHPLC Pump, Thermo Scientific Vanquish UHPLC Column Compartment, Thermo Scientific TSQQuantiva coupled to Thermo Scientific Column: Acuity UPLC BEH C18 1.7 μM, 2.1 x 100 mm Buffer A: 0.1% CH2O2 in H2O Buffer B: 0.1% CH2O2 in C2H3N Flow gradient [Table 1] Autosampler parameters: Withdrawal speed: 0.5μl / sec Dispensing speed: 5.0 μl / sec Column chamber: Operating temperature control: Check Temperature (℃):50.0

[0086] MS parameters Ion source type: H-ESI Spraying voltage: static Polarity: Positive Ion transfer tube temperature (℃): 275 Vaporizer temperature (℃): 325 Residence time (ms): 50 Use calibrated RF lens: Check Q1 resolution (FWHM): 0.7 Q3 resolution (FWHM): 0.7 CID gas (mTorr): 2 The spray voltage and collision energy (V) are analyte specific.

[0087] Data analysis Data analysis was performed using Thermo Scientific's Chromatography Data System Software "Chromeleon 7" (version 7.3.1 CDS). Fragmentation efficiency = product area under the curve / precursor area under the curve * 100

[0088] The quantitative ions based on the individual (poly)labels of formula (I) were as listed in Table 1 below. [Table 2]

[0089] Reference Example 3: Derivatization of tryptic peptides at the N-terminus Tryptic peptides with the following peptide sequences (N-terminal to C-terminal sequences) were derivatized: Peptide 1 ATNSQFLR (SEQ ID NO: 1) Peptide 2 FSPDDSAGASALLR (SEQ ID NO: 2) Peptide 3 VIFDANAPVAVR (SEQ ID NO: 3)

[0090] Derivatization at the N-terminus of the peptides was carried out with the following carbamate-based (poly)labeling tags: 1)DSS-UUUU*Prg*Prg**NH2 2)DSS-UUUU*Prg*Prg*Prg*Prg**NH2 where "DSS" represents the remainder of disuccinimidyl suberate with the remaining NHS ester group, "U" represents the remainder of 3-aminopropanoic acid, and "Prg*" represents the remainder of propargylglycine and N3 + -(CH2)3-OC(=O)-NH-(CH2)2-N + The structure of (poly)labeled tags (1) and (2) is shown below: [ka] [ka]

[0091] Synthesis of (poly)labeled tags a) Synthesis of H-UUUU-PrgPrgNH2 or H-UUUUPrgPrgPrgPrgNH2 Peptides were synthesized by fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis on a peptide synthesizer (e.g., Protein Technologies, Inc.). Five equivalents of each amino acid derivative (Fmoc-propargyl-glycine and Fmoc-beta-alanine) were used for amino acid coupling. The amino acid derivatives were dissolved in dimethylformamide containing 1 equivalent of 1-hydroxy-7-azabenzotriazole (HOAt). Peptides were synthesized on Tentagel® resin. The coupling reaction was carried out for 5 minutes in dimethylformamide containing 5 equivalents of HATU and 10 equivalents of N,N-diisopropylethylamine relative to the resin loading. The Fmoc group was cleaved for 8 minutes after each synthesis step using 20% ​​piperidine in dimethylformamide. Release of the peptide from the resin was achieved by incubation with 95% TFA, 2.5% triisopropylsilane, and 2.5% water for 3 hours. The peptide was then precipitated by mixing the reaction solution with chilled diisopropyl ether. The precipitate was filtered, rinsed again with diisopropyl ether, dissolved in a small amount of aqueous acetic acid, and lyophilized. The resulting crude material was purified by preparative RP-HPLC using an acetonitrile / water gradient containing 0.1% trifluoroacetic acid. The identity of the purified material was confirmed by ion spray mass spectrometry.

[0092] b) Attachment of labeling groups: Synthesis of H-UUUU*Prg*Prg**NH2 or H-UUUU*Prg*Prg*Prg*Prg**NH2 The alkyne-containing peptide prepared in (a) (1 equivalent) and an azide-containing label (4.4 equivalents), e.g., N3 + -(CH2)3-OC(=O)-NH-(CH2)2-N +(CH3)3 was dissolved in water / acetonitrile (1:1) and a solution of CuBr (1.5 eq., 0.1 M in acetonitrile), THPTA (1.5 eq., 0.1 M in water), and trimethylamine (3 eq.) was added. The reaction mixture was kept under argon atmosphere and shaken (700 rpm) at 32 °C. After 20 h, a solution of EDTA (pH 8.0) was added and the mixture was shaken at room temperature for 10 min. The solution was then diluted with water (1:1), filtered, and purified by flash chromatography on a C-18 column. Product-containing fractions were pooled and lyophilized. The identity of the product was confirmed by LC-MS and HPLC. 1 This was verified by H-NMR.

[0093] c) Attachment of DSS to (poly)labeled peptides To a solution of disuccinimidyl suberate (DSS) (1 equivalent) in dry DMF was added the peptide (1.3 equivalents) obtained in (b) dissolved in DMF and diisopropylamine (2 equivalents) and stirred at room temperature for 2 hours. The product was purified by preparative HPLC. The identity of the purified material was confirmed by ion spray mass spectrometry.

[0094] For derivatization at the N-terminus of the peptide, 30 μM peptide was reacted with 150 μM of the (poly)labeled tag (1) or (2) obtained from (c) in 100 mM aqueous sodium bicarbonate solution at pH 8.0 at 37°C for 3 hours.

[0095] Reference Example 4: Synthesis of tryptic peptides with a (poly)tag at the C-terminus Peptide 1 (ATNSQFLR, SEQ ID NO: 1) was added to Prg ## NH2 [(poly)labeling tag (3)] was derivatized. ## NH2” is linked to N via its alkenyl group (via click chemistry) 3+ represents the remainder of the reaction product of propargylglycine coupled with a -Z group, where Z is a nucleoside having adenine as the base, and the terminal COOH group of propargylglycine is amidated.

[0096] Peptide 1 (ATNSQFLR, SEQ ID NO: 1) was also synthesized by the Prg # Prg ## The poly((poly)-labeled tag (4) was derivatized with NH2. #」 is linked to N via its alkenyl group (via click chemistry) 3+ represents the remainder of the reaction product of propargylglycine coupled with a -Z group, where Z is a nucleoside having adenine as the base, and a terminal "Prg ## "NH" indicates that the terminal COOH group of the final propargylglycine is amidated. The resulting structure is shown below. [ka]

[0097] Comparative Example 1: MS / MS spectrum of peptide ATNSQLFR without (poly)labeling The selective fragmentation of peptide 1 ATNSQFLR (SEQ ID NO: 1) was investigated by MS / MS, and the respective MS / MS spectra are shown in Figure 1.

[0098] Under collision-induced dissociation (CID) conditions in the mass spectrometer, the peptides collided with an inert gas and, as usual, broke at one weak bond, typically an amide bond, to produce peptide fragment ions ( Figure 1 ), resulting in a large number of fragment ions, as shown in Figure 1. This limited the detection sensitivity of peptides, as the signal was split into many fragment ions, as shown in Figure 1 .

[0099] Example 1: Selective fragmentation to generate quantitative ions—MS / MS spectra of peptides with a C-terminal adenine tag Peptide 1a, which contains peptide 1 (ATNSQFLR, SEQ ID NO: 1) with a single adenine-containing polylabel attached to the C-terminus, prepared according to Reference Example 4, was examined by MS / MS. The MS / MS spectrum is shown in Figure 2. Unlike the peptide without a polylabel (Comparative Example 1, see Figure 1), the peptide polylabel construct with an adenine-containing polylabel as the label was shown to be selectively resolved, generating a highly abundant quantitation ion (136 Da).

[0100] Example 2: Selective fragmentation to generate quantitative ions—MS / MS spectra of peptides with an APG label at the C-terminus Peptide 1 ATNSQFLR (SEQ ID NO: 1), which has a blocked (acetylated) N-terminus, was derivatized with an APG-containing (poly)label at the C-terminus, i.e., with an APG group coupled to the free COOH group of the glutamic acid side chain by an amide bond, and the C-terminus of the final glutamic acid was amidated. A glutamic acid was coupled to the C-terminus of peptide 1 and examined via MS / MS. The MS / MS spectrum is shown in Figure 3. Unlike the peptide without a (poly)label (Comparative Example 1, see Figure 1), the APG-based peptide (poly)label was shown to generate highly abundant "quantitative ions" (PG, proline-glycine, MH). +1 =172Da).

[0101] Example 3: Selective fragmentation to generate quantitative ions—MS / MS spectra of peptides with carbamate-based labels at the C-terminus Peptide 1 ATNSQFLR (SEQ ID NO: 1) with one carbamate containing (poly)label (2) attached via an amide bond to the C-terminus, prepared according to Reference Example 3, was examined by LCMS / MS. The MS / MS spectrum is shown in Figure 4.

[0102] Unlike the peptide without a (poly)label (Comparative Example 1, see Figure 1), the labile chemical analyte (HO-CO-NH-CH-CH-N of 147 Da) +(CH3)3) decomposes from the peptide-(poly)label construct, followed by water loss to form MH +1 It was shown that a stable quantitative ion of 129 Da was generated (O=CNH-CH-CH-N + (CH3)3).

[0103] Example 4: Multiple fragmentation events in individual analytes - adenine-based (poly)labeling at the C- and N-termini Peptide (poly)-labeled constructs with multiple copies of the "quantitation moiety" were expected to undergo multiple fragmentation events on each individual peptide. To demonstrate this, peptide 1 ATNSQFLR (SEQ ID NO: 1), coupled with a C-terminal (poly)-tag and an N-terminal (poly)-tag, was synthesized according to Reference Example 4 and analyzed by LC-MS / MS. The MS / MS spectrum is shown in Figure 5. Because this peptide (poly)-labeled construct had adenine-based (poly)-tags at each end, detection of peptide (poly)-labeled fragments lacking both "quantitation moieties" indicated that multiple fragmentation events had occurred on each individual peptide (poly)-labeled construct.

[0104] Example 5: Multiple fragmentation events in individual analytes - APG-based (poly)labeling at the C-terminus and N-terminus Peptide (poly)labeled constructs with multiple copies of a particular "moiety" were expected to undergo multiple fragmentation events on each individual peptide. To demonstrate this, peptide 1 ATNSQFLR (SEQ ID NO: 1) was derivatized with one APG-containing (poly)label at the N-terminus and one APG-containing (poly)label at the N-terminus. That is, two glutamic acids, each with an APG group coupled to the free COOH group of the glutamic acid by an amide bond, were coupled and analyzed by LC-MS / MS. The MS / MS spectrum is shown in Figure 6. Because this peptide (poly)labeled construct had two APG-based (poly)labels, detection of peptide (poly)labeled fragments lacking both "quantitation moieties" indicated that multiple fragmentation events had occurred on each individual peptide (poly)labeled construct.

[0105] Example 6: Multiple fragmentation events in individual analytes - Carbamate-based (poly)labeling at the C- and N-terminus Peptide (poly)-labeled constructs with multiple copies of the "quantitation moiety" were expected to undergo multiple fragmentation events on each individual peptide. To demonstrate this, peptide 1 ATNSQFLR (SEQ ID NO: 1), which has a (poly)tag with carbamate residues at the C-terminus and N-terminus [(poly)-labeled tag (1)], was synthesized according to Reference Example 3 and analyzed by LC-MS / MS. The MS / MS spectrum is shown in Figure 7. Because this peptide (poly)-labeled construct had two carbamate residues, detection of peptide (poly)-labeled fragments lacking both "quantitation moieties" indicated that multiple fragmentation events had occurred on each individual peptide (poly)-labeled construct.

[0106] Example 7: Signal intensity or fragmentation efficiency increases with multiple adenine-based (poly)labels Peptide 1 ATNSQFLR (SEQ ID NO: 1) with one adenine residue attached to the C-terminus was synthesized based on the (poly)label tag (3) according to Reference Example 4, and polypeptide ATNSQFLR (SEQ ID NO: 1) with two adenine residues attached to the C-terminus was also synthesized based on the (poly)label tag (4) according to Reference Example 4. The results were analyzed based on LC-MS / MS data in terms of comparing relative selected reaction monitoring (SRM) intensities. The results are shown graphically in Figure 8. Relative SRM strength = SRM strength (未改変ペプチド) / SRM strength (Pep.~n((ポリ)標識))

[0107] The presence of multiple units of "moieties" and the highly selective fragmentation of (poly)label-containing peptides were shown to increase the SRM intensity of the quantitation ions. Furthermore, as the number of (poly)labels per analyte increased, the relative fragmentation efficiency was shown to increase, with multiple fragmentation events per individual peptide molecule, as shown in Figure 10. Fragmentation efficiency (%) = (product ion intensity / precursor ion intensity) * 100 Relative fragmentation efficiency = fragmentation efficiency (Pep.~n((ポリ)標識)) / Fragmentation efficiency (未改変ペプチド)

[0108] Example 8: Signal intensity or fragmentation efficiency increases with multiple APG-based (poly)labels Peptide 1 ATNSQFLR (SEQ ID NO: 1), which has one APG residue attached to its C-terminus, and peptide 1 ATNSQFLR (SEQ ID NO: 1), which has two APG residues attached to its C-terminus, were investigated based on LC-MS / MS data in terms of comparing their relative selected reaction monitoring (SRM) intensities. The results are shown graphically in Figure 9. Relative SRM strength = SRM strength (未改変ペプチド) / SRM strength (Pep.~n((ポリ)標識))

[0109] The presence of multiple units of "moieties" and the highly selective fragmentation of (poly)label-containing peptides were shown to increase the SRM intensity of the quantitation ions. Furthermore, as the number of (poly)labels per analyte increased, the relative fragmentation efficiency was shown to increase, with multiple fragmentation events per individual peptide molecule, as shown in Figures 10 and 11. Fragmentation efficiency (%) = (product ion intensity / precursor ion intensity) * 100 Relative fragmentation efficiency = fragmentation efficiency (Pep.~n((ポリ)標識)) / Fragmentation efficiency (未改変ペプチド)

[0110] Example 9: Investigation of a greater number of quantification moieties per analyte 9a: Relative SRM signal intensities of carbamate-based (poly)labels To investigate the feasibility of accommodating multiple (>2) polylabels per analyte, carbamate-based 1x, 2x, 4x, and 6x polylabel-peptide constructs were synthesized and investigated by LCMS. The resulting relative selected reaction monitoring (SRM) intensities were compared. The results are shown graphically in Figure 12. Relative SRM strength = SRM strength (未改変ペプチド) / SRM strength (Pep.~n((ポリ)標識))

[0111] In this example, approximately 2.5-fold higher SRM intensities were measured using the peptide-6x((poly)labeled) construct.

[0112] 9b: Relative fragmentation efficiencies of carbamate-based (poly)labels The relative fragmentation efficiencies were calculated for 1×, 2×, 4× and 6× (poly)labeled peptide constructs based on the carbamate of 9a: Fragmentation efficiency (%) = (product ion intensity / precursor ion intensity) * 100 Relative fragmentation efficiency = fragmentation efficiency (Pep.~n((ポリ)標識)) / Fragmentation efficiency (未改変ペプチド)

[0113] The results are shown graphically in Figure 13. Unlike the adenine and APG-based (poly)labels, the carbamate-based (poly)labels only resulted in a slight increase in fragmentation efficiency.

[0114] Example 10: Derivatization of tryptic peptides with NHS esters containing 2x and 4x (poly)labels To demonstrate the applicability of the (poly)labeling concept to tryptic peptides, three different synthetic tryptic peptides (peptide 1 of SEQ ID NO: 1, peptide 2 of SEQ ID NO: 2, and peptide 3 of SEQ ID NO: 3) were reacted at their N-termini with NHS-ester reactive groups containing carbamate-based 2x- and 4x-(poly)tags [(poly)labeled tag (1), (poly)labeled tag (2), see Reference Example 3], and after derivatization, the peptides were analyzed by LCMS. The relative fragmentation efficiencies were calculated: Relative fragmentation efficiency = fragmentation efficiency (Pep.-n~4x((ポリ)標識)) / Fragmentation efficiency (Pep.-n~2x((ポリ)標識))

[0115] The results are shown graphically in Figure 14. For all three peptides, the 4x (poly)labeled peptide construct resulted in nearly four times the fragmentation efficiency. It is highly plausible to expect that the four-fold higher fragmentation efficiency would also be reflected in the SRM intensity in the case of 100% derivatization of the peptide.

[0116] References -Hahne et al.2013:Hahne,H.,Pachl,F.,Ruprecht,B.et al.DMSO enhances electrospray response,boosting sensitivity of proteomic experiments.Nat Methods 10,989-991(2013) -Mirzaei et al.2006:Mirzaei,H.;Regnier,F.Enhancing electrospray ionization efficiency of peptides by derivatization.Anal.Chem.2006,78,4175-4183

Claims

1. 1. Use of a (poly)label to generate a quantifiable signal for an analyte of interest in mass spectrometry, said (poly)label having the structure (I): 【Chemistry 1】 During the ceremony, m is 0 or 1; n is 0 or an integer selected from the range of 1 to 20; Q is absent or a linker unit; X is a reactive group or, if Q is absent, a hydrogen atom; Y is an amino acid based linker unit having a side chain suitable for coupling to Z; Y １ is a linker unit based on an amino acid having a side chain suitable for coupling to Z, said amino acid having a blocked carboxylic acid group or a blocked amino group; Z is a group consisting of: - nucleosides comprising a nucleobase selected from the group consisting of adenine, cytosine, thymine, guanine and uracil: -Structure Z １ -Proline-Z ２ A tripeptide of the formula: １ and Z ２ are, independently of each other, amino acids selected from the group consisting of alanine, glycine, valine, leucine, iso-leucine and phenylalanine; and -Carbamate group -O-C(=O)-NH-(CH ２ ) ｚ -N ＋ (Z ３ ) ３ or —NH—C(═O)—O—(CH ２ ) ｚ -N ＋ (Z ３ ) ３ wherein z is an integer selected from the range of 1 to 10; Z ３ is a C1-C5 alkyl group.

2. 2. The use according to claim 1, wherein the (poly)label has the structure of formula (Ia): 【Chemistry 2】 During the ceremony, n is an integer selected from the range of 1 to 20; Q is absent or a linker unit; X is a reactive group; Y is, 【Transformation 3】 Base, or 【Chemistry 4】 a group wherein the dotted line at the oxygen atom indicates the bond to Z and u is either 1 or 2, or 【Transformation 5】 Base, or 【Transformation 6】 basis, or 【Transformation 7】 Base, or 【Transformation 8】 a group wherein the dotted line at the N or C atom in the triazole ring represents the bond to Z, t is 0 or 1; v. 1 is 0 or an integer ranging from 1 to 4; v. 2 is an integer ranging from 1 to 10; w is 0 or 1; R １ is a hydrogen atom or a C1-C5 linear or branched alkyl group; R ２ , R ３ are independently selected from a hydrogen atom and a C1 to C5 straight or branched alkyl group; R ４ is a hydrogen atom or a C1-C5 linear or branched alkyl group; R ５ is a hydrogen atom; R ６ is absent or is a —C(═O)NH— group or a C(═)O— group or a substituted or unsubstituted C6-C10 arylene, wherein one or more substituents are selected from a hydrogen atom, a halogen atom, and a functional group; R ７ is absent or selected from the group consisting of branched or unbranched C1-C5 alkylene, —O—C1-C5 alkylene, wherein said C1-C5 alkylene is branched or unbranched, and —S—C1-C5 alkylene, wherein said C1-C5 alkylene is branched or unbranched; Or, R ４ and R ５ together form NR as part of a ring structure. ４ forming a 5- or 6-membered heteroalkyl ring containing a nitrogen atom of the formula: NH or NR for each Y group ４ The dotted line in indicates Q, the next [Y-Z] unit, or Y １ The dotted line at the C(=O) of each group represents the bond to the next [Y-Z] unit, Y １ or a bond to Q; Y １ teeth, 【Chemistry 9】 Base or 【Chemistry 10】 a group wherein the dotted line at the oxygen atom indicates the bond to Z and u is either 1 or 2, or 【Chemistry 11】 Base, or 【Chemistry 12】 basis, or 【Chemistry 13】 Base, or 【Chemistry 14】 basis, or 【Chemistry 15】 Base, or 【Chemistry 16】 basis, or 【Chemistry 17】 Base, or [Chemistry 18] It is the basis, In the formula, the dotted line at the N atom or C atom in the triazole ring represents the bond to Z, and t, v.1, v.2, w and R １ ~R ５ has the same meaning as above for Y; Each Y １ NH or NR group ４ or the dotted line at the C(=O) represents the bond to the next [Y-Z] unit; Z is a group consisting of: - a nucleoside having a nucleobase selected from the group consisting of adenine, cytosine, thymine, guanine and uracil; and -Carbamate group -O-C(=O)-NH-(CH ２ ) ｚ -N ＋ (Z ３ ) ３ or —NH—C(═O)—O—(CH ２ ) ｚ -N ＋ (Z ３ ) ３ wherein z is an integer selected from the range of 1 to 20; Z ３ is a C1-C5 alkyl group; and -Tripeptide Z １ -Proline-Z ２ [In the formula, Z １ and Z ２ are, independently of one another, amino acids selected from the group consisting of alanine, glycine, valine, leucine, iso-leucine and phenylalanine; Z ２ wherein the C-terminus is preferably blocked, more preferably amidated.

3. 3. The use according to claim 1 or 2, wherein the (poly)label has a structure of formula (Ia), wherein n is an integer selected from the range of 1 to 20; X is a hydrogen atom; Q is absent; Y is, 【Chemistry 19】 group, or 【Chemistry 20】 basis, or 【Chemistry 21】 group, or 【Chemistry 22】 is a group; In the formula, NR ４ The dotted line in represents a bond to Q or the next [Y-Z] unit, respectively, and the dotted line in C(=O) represents a bond to the next [Y-Z] unit or Y １ The dotted line at the N atom or C atom in the triazole ring represents a bond to Z, and t, v.1, v.2, w and R １ ~R ５ has the same meaning as given above in claim 2; Y １ teeth, 【Chemistry 23】 group, or 【Chemistry 24】 basis, or 【Chemistry 25】 group, or 【Chemistry 26】 basis, or 【Chemistry 27】 group, or 【Chemistry 28】 basis, or 【Chemistry 29】 group, or 【Transformation 30】 is a group; In the formula, NR ４ or C(═O) represents a bond to the next [Y-Z] unit, the dotted line at the N atom or C atom in the triazole ring represents a bond to Z, and t, v.1, v.2, w and R １ ~R ５ has the same meaning as given above in claim 2; and Z is a nucleoside having a nucleobase selected from the group consisting of adenine, cytosine, thymine, guanine and uracil.

4. 3. The use according to claim 1 or 2, wherein the (poly)label has a structure of formula (Ia), wherein n is an integer selected from the range of 1 to 20; Q is a linker unit, and X is a reactive group; Y is, 【Chemistry 31】 group, or 【Chemistry 32】 basis, or 【Transformation 33】 group, or 【Transformation 34】 It is the basis, In the formula, NR ４ The dotted line in represents a bond to Q or the next [Y-Z] unit, respectively, and the dotted line in C(=O) represents a bond to the next [Y-Z] unit or Y １ The dotted line at the N atom or C atom in the triazole ring represents a bond to Z, and t, v.1, v.2, w and R １ ~R ５ has the same meaning as given above in claim 2; Y １ teeth, 【Chemistry 35】 group, or 【Transformation 36】 basis, or 【Chemistry 37】 group, or 【Transformation 38】 basis, or 【Chemistry 39】 group, or 【Chemistry 40】 basis, or 【Chemistry 41】 group, or 【Chemistry 42】 is a group; In the formula, NR ４ or C(═O) represents a bond to the next [Y-Z] unit, the dotted line at the N atom or C atom in the triazole ring represents a bond to Z, and t, v.1, v.2, w and R １ ~R ５ has the same meaning as given above in claim 2; and Z is a carbamate group -O-C(=O)-NH-(CH ２ ) ｚ -N ＋ (Z ３ ) ３ or —NH—C(═O)—O—(CH ２ ) ｚ -N ＋ (Z ３ ) ３ wherein z is an integer selected from the range of 1 to 10; Z ３ is a C1 to C5 alkyl group.

5. 3. The use according to claim 1 or 2, wherein the (poly)label has a structure of formula (Ia), wherein n is an integer selected from the range of 1 to 20; Q is absent or is a linker unit; X is a hydrogen atom or a reactive group; Y is, 【Chemistry 43】 group, where the dotted line at the oxygen atom indicates the bond to Z, u is 1 or 2, the dotted line at NH represents the bond to Q or the next [Y-Z] unit, respectively, and the dotted line at C(=O) represents the bond to the next [Y-Z] unit or Y, respectively. １ represents a bond to Y １ teeth, 【Chemistry 44】 group, or 【Chemistry 45】 It is the basis, wherein the dotted line at the oxygen atom represents the bond to Z, u is 1 or 2, and the dotted line at the NH or C(═O) represents the bond to the next [Y—Z] unit; and Z is tripeptide Z １ -Proline-Z ２ wherein Z １ and Z ２ are, independently of one another, amino acids selected from the group consisting of alanine, glycine, valine, leucine, iso-leucine and phenylalanine; Z ２ The C-terminus of is preferably blocked, more preferably amidated.

6. 3. The use according to claim 1 or 2, wherein in the (poly)label structure of formula (I), n and m are both 0, Q is absent, and X is a hydrogen atom, and the (poly)label has the structure of formula (Ib): Z １ -Proline-Z ２ (Ib) During the ceremony, Z １ and Z ２ are, independently of one another, amino acids selected from the group consisting of alanine, glycine, valine, leucine, iso-leucine and phenylalanine; Z ２ The C-terminus of is preferably blocked, more preferably amidated.

7. Q is (C1-C5 alkylene-O-) ｒ a unit [r is an integer ranging from 1 to 10] or a C1-C20 alkanediyl unit or a C1-C20 alkanediyl-heteroaryl unit or a (C1-C5 alkanediyl)-O-(C1-C5 alkanediyl) unit or a C2-C9 alkanediyl-C(=O) unit or a C2-C9 alkanediyl-NH unit, preferably a C4-C7 alkanediyl-C(=O) unit or a C4-C7 alkanediyl-NH unit, more preferably a hexylene-C(=O) unit or a hexylene-NH unit, or [NH-C1-C5 alkanediyl-C(=O)] ｘ unit or [C(═O)—C1-C5 alkanediyl-NH] ｘ 7. The use according to any one of claims 1 to 6, wherein the linker comprises a unit wherein x is an integer selected from the range of 1 to 20; and / or the C1-C5 alkanediyl is preferably a C2-C4 alkanediyl, more preferably ethylene.

8. 8. The use according to any one of claims 1 to 7, wherein X is a reactive group selected from the group consisting of an isothiocyanate group, an isocyanate group, an acyl azide group, a sulfonyl chloride group, an aldehyde group, a glyoxal group, an epoxide group, an oxirane group, a carbonate group, an aryl halide group, an imidoester group, a carbodiimide group, an anhydride group, a fluorophenyl ester group, a carboxyl group, a HATU ester group, an HBTU ester group and an NHS ester group, preferably an NHS ester group.

9. 1. A process for modifying an analyte of interest to obtain an increased intensity signal in mass spectrometry, comprising the steps of: (a) providing at least one (poly)label having a reactive group of structure (I), 【Chemistry 46】 During the ceremony, m is 0 or 1; n is 0 or an integer selected from the range of 1 to 20; Q is absent or a linker unit; X is a reactive group or, if Q is absent, a hydrogen atom; Y is an amino acid based linker unit having a side chain suitable for coupling to Z; Y １ is a linker unit based on an amino acid having a side chain suitable for coupling to Z, said amino acid having a blocked carboxylic acid group or a blocked amino group; Z is a group consisting of: - nucleosides comprising a nucleobase selected from the group consisting of adenine, cytosine, thymine, guanine and uracil: -Structure Z １ -Proline-Z ２ A tripeptide of the formula: １ and Z ２ are, independently of each other, amino acids selected from the group consisting of alanine, glycine, valine, leucine, iso-leucine and phenylalanine; and -Carbamate group -O-C(=O)-NH-(CH ２ ) ｚ -N ＋ (Z ３ ) ３ or —NH—C(═O)—O—(CH ２ ) ｚ -N ＋ (Z ３ ) ３ wherein z is an integer selected from the range of 1 to 10; Z ３ is a C1-C5 alkyl group; (b) providing an analyte of interest, the analyte being selected from the group consisting of polypeptides and small molecules, preferably a polypeptide having free amino and / or free carboxyl groups, which, if present, are optionally activated; (c) reacting said at least one (poly)label having a reactive group with said analyte of interest, thereby obtaining a reaction product, wherein said at least one (poly)label is covalently bound to said analyte of interest.

10. 1. A method for determining an analyte of interest by mass spectrometry, comprising: (i) providing a reaction product of said analyte of interest, said reaction product being based on a (poly)label having a structural element (I), 【Chemistry 47】 During the ceremony, covalently attached to said analyte of interest, wherein Q, X, Y, Y １ , Y ２ providing a reaction product, wherein Z, m and n have the meanings defined in any one of claims 1 to 8; (ii) subjecting the reaction product provided in (i) to mass spectrometry; (iii) (unsubstituted) MH of Z ＋１ determining the intensity in the mass spectrum of the fragment corresponding to the peak or its water depletion product.

11. A (poly)label having the structure of formula (I), 【Chemistry 48】 In the formula, Q, X, Y, Y １ , Y ２ , Z, n and m have the meanings defined in any one of claims 1 to 8. (Poly)label.

12. A reaction product comprising a polypeptide and a (poly)label having the general structural formula (III): 【Chemistry 49】 In the formula, Q, X, Y, Y １ , Y ２ , Z, n and m have the meanings defined in any one of claims 1 to 8, and the designations x and y are either 0 or 1, with the proviso that at least one of x and y is 1, and R is the remainder of said polypeptide.

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