Derivative agents for laser desorption ionization mass spectrometry

Abstract

In a first aspect, the invention relates to a derivatizing agent, preferably a derivatizing agent for an analyte intended for analysis via LDI-MS, said derivatizing agent comprising a structural element of formula (I) C-L1-Z-(L2) p-X wherein C is a chromophore having an absorption maximum in the range of 280 to 400 nm; z is a charged unit comprising at least one permanently charged portion; x is a reactive group; l1 and L2 are respectively a joint unit; and p is zero or 1. A second aspect of the invention relates to a kit comprising the derivatizing agent according to the first aspect. In a third aspect, the invention relates to the use of a derivatizing agent according to the first aspect for the mass spectrometry of analyte molecules wherein the mass spectrometry is LDI-MS. A fourth aspect of the invention relates to a conjugate of a derivatizing agent and an analyte according to the first aspect wherein the conjugate has the structure of formula (II) C-L1-Z-(L2) p-Xa-Ya-A wherein C, L1, L2, p, Z and N are as defined in the context of the first aspect; xa is the remainder of the reactive group X as defined in the context of the first aspect; a is an analyte and Ya is the remainder of the reactive group Y bound to the analyte A, which has reacted with the reactive group X of the derivatizing agent, thereby forming a covalent bond between Xa and Ya. A fifth aspect of the invention relates to a method for the mass spectrometry of analyte molecules, comprising the steps of: (a) providing a target analyte; (b) providing a derivatizing agent comprising the structure of formula (I) as defined in the context of the first aspect; (c) reacting the analyte provided according to (a) with the derivatizing agent provided according to (b), thereby forming a conjugate of the analyte and the derivatizing agent; and (d) subjecting the conjugate formed in (c) to mass spectrometric analysis, wherein the mass spectrometric analysis is preferably LDI-MS.

Description

[0001] In a first aspect, the present invention relates to a derivatizing agent, preferably for an analyte intended for analysis by LDI-MS, the derivatizing agent comprising a structural element of formula (I) C–L1–Z–(L2) p –X, wherein C is a chromophore having an absorption maximum in the range from 280 to 400 nm; Z is a charged unit comprising at least one permanently charged moiety; X is a reactive group; L1 and L2 are each a linker unit; and p is zero or 1.

[0002] A second aspect of the present invention relates to a kit comprising the derivatizing agent according to the first aspect. In a third aspect, the present invention relates to the use of the derivatizing agent according to the first aspect for mass spectrometry of analyte molecules, wherein the mass spectrometry is LDI-MS. A fourth aspect of the present invention relates to a conjugate of the derivatizing agent according to the first aspect and an analyte, wherein the conjugate has the structure of formula (II) C–L1–Z–(L2) p –Xa–Ya–A, wherein C, L1, L2, p, Z and N are as defined in the context of the first aspect; Xa is the remainder of the reactive group X as defined in the context of the first aspect; A is the analyte and Ya is the remainder of the reactive group Y bound to the analyte A, which has reacted with the reactive group X of the derivatizing agent, thereby forming a covalent bond between Xa and Ya. A fifth aspect of the present invention relates to a method for mass spectrometry of analyte molecules, the method comprising the steps of: (a) providing a target analyte; (b) providing a derivatizing agent comprising a structure of formula (I) as defined in the context of the first aspect; (c) reacting the analyte provided according to (a) with the derivatizing agent provided according to (b), thereby forming a conjugate of the analyte and the derivatizing agent; and (d) subjecting the conjugate formed in (c) to mass spectrometry, wherein the mass spectrometry is preferably LDI-MS. Background Art

[0003] Mass spectrometry (MS) is a technique widely used in the qualitative and quantitative analysis of chemical substances from small to large molecules. Generally, it is a very sensitive and specific method, even allowing the analysis of complex biological samples (such as environmental or clinical samples). However, for several analytes, especially if analyzed from complex biological matrices such as serum, the sensitivity of the measurement remains a problem.

[0004] MS is often coupled with chromatographic techniques, especially gas chromatography and liquid chromatography such as HPLC for example. Here, the target molecules to be analyzed are chromatographically separated and mass spectrometric analysis is performed separately (Higashi et al., (2016) J. of Pharmaceutical and Biomedical Analysis 130, pp. 181-190).

[0005] However, there is still a need to increase the sensitivity of MS analysis methods, especially for analyzing analytes with low abundance or when only extremely small amounts of material (such as biopsy tissue) are available.

[0006] In the art, several derivatization reagents aimed at improving the measurement sensitivity of these analytes are known. Among them, reagents that contain a charged unit and a neutral loss unit combined in a single functional unit (e.g., WO 2011 / 091436 A1); other reagents for introducing a neutral loss unit and a charged unit are known, for example from WO2020 / 020850 A1. Other reagents containing independent units are relatively large in structure, which affects the general workflow of sample preparation and MS measurement (Rahimoff et al. (2017) J. Am. Chem. Soc. 139(30), pp. 10359-10364). Known derivatization reagents are for example Cookson type reagents, Amplifex Diene, Amplifex Keto, Girard T, Girard P. All of these reagents have drawbacks in that there is usually insufficient labeling efficiency, the coupling chemical reaction generates structural isomers, poor ionization efficiency, poor chromatographic separation after coupling, poor fragmentation behavior due to many fragmentation pathways, and the need for high collision energy. Therefore, there is an urgent need in the art for derivatization reagents that allow for the sensitive detection of analytes from complex biological matrices and exhibit a chemical structure that does not negatively impact the MS measurement workflow. This is particularly important in random access high-throughput MS setups, where several different analytes with different chemical properties have to be measured within a short time.

[0007] Chemical derivatization of target analytes can be used to improve detection sensitivity in mass spectrometry applications. In most cases, charged (i) or chargeable (ii) compounds are used to produce (i) permanently charged or (ii) chargeable derivatized analytes to improve mass spectrometry response / sensitivity. Most of these reagents are designed to enhance the ESI response, but they are not designed to generate specific product ions for MS / MS applications by CID. To address this issue, several derivatization reagents have been developed that have structures suitable for MS / MS detection. Generally, many derivatization reagents can be used for liquid chromatography-based mass spectrometry (i.e., LC-MS, LC-MS / MS; see reviews: J. Sep. Sci. 2016, 39, 102–114; Biomed. Chromatogr. 2011; 25:1–10) and combinations with suitable matrices for laser desorption-based applications (i.e., MALDI, see reviews: Trends in Analytical Chemistry 143 (2021) 116399; J. Mass Spectrom. 2021; 56: e4731). However, a large number of available derivatization reagents do not possess suitable chromophores for efficient laser energy transfer in LDI applications and are thus not suitable for high-sensitivity LDI measurements. Therefore, these reagents need to be used in combination with matrices, which results in increased background, interference, and thus reduced sensitivity. Only a few examples of derivatization reagents for matrix-free laser desorption applications have been reported, which are referred to as “reactive matrices” (i.e., Anal. Chem. 2020, 92, 6224–6228; Chem Asian J. 2021, 16, 868–878 and Crit. Rev. Anal. Chem. 2021 Dec 30; 1–17) or “LDI tags” (i.e., Mass Spec Rev. 2019; 38:3–21; Scientific Reports | 5:17853, ChemBioChem 2021, 22, 1430–1439;). Most of these molecules provide suitable chromophores but are not permanently charged, which is disadvantageous in terms of high sensitivity and are not designed for MS / MS applications. Rare examples of permanently charged and chromogenic derivatization reagents have been reported (i.e., International Journal of Mass Spectrometry 353 (2013) 54–59), but these reagents lack suitable neutral loss sites and are thus not suitable for MS / MS applications. In most cases, derivatization reagents are small molecules, and in cases where the analyte is also a small molecule, low-molecular-weight derivatized analytes are produced, or in any case, only small mass shifts occur after derivatization.This can be problematic for several mass spectrometry applications, since high-sensitivity measurements in the lower molecular weight range are typically hampered by matrix-based MS interferences in that range.

[0008] Accordingly, the problem underlying the present invention is the need for permanently charged and chromophoric derivatization reagents having suitable neutral loss sites.

[0009] Aspect 1 – derivatizing agent

[0010] In a first aspect, this problem is solved by a derivatizing agent, preferably for derivatizing analytes intended to be analyzed via laser desorption ionization mass spectrometry (LDI-MS), which derivatizing agent comprises a structural element of formula (I)

[0011] C–L1–Z–(L2) p –X (I)

[0012] wherein

[0013] C is a chromophore having an absorption maximum in the range from 280 to 400 nm;

[0014] Z is a charged unit which comprises at least one permanently charged moiety;

[0015] X is a reactive group;

[0016] L1 and L2 are each a linker unit; and

[0017] p is zero or 1.

[0018] The derivatizing agent according to the present invention provides a solution for challenging high-sensitivity measurements in the field of mass spectrometry (MS) based on laser desorption ionization (LDI), preferably but not limited to. The advantage of this derivatizing agent is the improved sensitivity for LDI-MS applications. The term "LDI-MS" includes (MA)LDI-MS, preferably (MA)LDI-MS / MS and (SA)LDI-MS, preferably (SA)LDI-MS / MS, where "MALDI" (matrix-assisted laser desorption ionization) and "SALDI" (surface-assisted laser desorption ionization) are known and will also be explained in more detail in the part related to the third aspect of the present invention below. The derivatizing agent is characterized by its chemical structure concept and its working principle being different from other reagents / solutions known in the art. In one aspect, the derivatizing agent carries a suitable chromophore, which enables efficient energy transfer during LDI. In another aspect, the derivatizing agent adds a sufficiently large molecular weight to the target analyte, where the weight addition causes a sufficient mass shift beyond the high-noise background in the low molecular weight region of the biological sample. Generally, such a high molecular weight is known to result in unfavorable ionization characteristics (poor ionization efficiency, multiple fragmentation processes...), however, in this instance, this is circumvented by the presence of a permanent positive charge (the Z unit in formula (I)). Thus, precursor ions (parent ions) can be detected with high sensitivity, and the precursor ions can optionally be selected for fragmentation in MS / MS applications. Effective fragmentation is ensured by a specific combination of the Z unit and the N unit, which is a quaternary ammonium group adjacent to the benzyl position in some embodiments outlined in more detail below, enabling a smooth and selective neutral loss fragmentation process. During MS / MS, the conjugate of the derivatizing reagent and the analyte fragments by releasing large fragments and producing ions (the target analyte modified with a benzyl cation). This large mass transfer is also very advantageous as it ensures a low background / interference for this novel class of reagents, since this specific and sensitive neutral loss pathway via large molecular weight loss is very rare (i.e., see Anal. Chem. 2014, 86, 21, 10724–10731).

[0019] Before the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methods, 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, the scope of which is limited only by the appended claims. Unless otherwise specified, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0020] The entire text of this specification incorporates by reference several documents. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions for use, etc.), whether cited above or below, is hereby incorporated by reference in its entirety. In the event of a conflict between the definitions or teachings of such incorporated references and the definitions or teachings cited in this specification, the text of this specification shall prevail.

[0021] The elements of the present invention will be described below. These elements are listed together with specific embodiments. However, it should be 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 present invention to only the specifically described embodiments. This description should be understood to support and cover embodiments that combine the specifically described embodiments with any number of the disclosed and / or preferred elements. In addition, unless the context otherwise indicates, any arrangement and combination of all the elements described in this application shall be considered to be disclosed by the specification of this application.

[0022] The word "comprise" and variations such as "comprises" and "comprising" should be understood to imply the inclusion of the 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.

[0023] As used in this specification and the appended claims, unless the context clearly dictates otherwise, the singular forms "a", "an", "the", and "said" include plural referents.

[0024] Percentages, concentrations, amounts, and other numerical data may be expressed or presented herein in a "range" format. It should be understood that such range formats are used merely for convenience and brevity and should therefore be interpreted flexibly to include not only the explicitly recited values as the limits of the range, but also all the individual values or sub-ranges subsumed within that range, as if each individual value and sub-range were explicitly recited. By way of illustration, the numerical range "4% to 20%" should be interpreted to include not only the explicitly recited values of 4% to 20%, but also the individual values and sub-ranges within the indicated range. Thus, the individual values such as 4%, 5%, 6%, 7%, 8%, 9%, 10%,... 18%, 19%, 20% and sub-ranges such as 4%-10%, 5%-15%, 10%-20%, etc. are included within the indicated numerical range. The same principle applies to ranges that recite a minimum or maximum value. In addition, such interpretation applies regardless of the breadth of the stated range or feature.

[0025] When used in connection with a numerical value, the term "about" is intended to cover numerical values within a range that has a lower limit that is 5% less than the indicated value and an upper limit that is 5% greater than the indicated value.

[0026] The term "Mass Spectrometry" ("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 typically involve (1) ionizing a compound to form a charged compound; and (2) detecting the molecular weight of the charged compound and calculating the mass-to-charge ratio. Compounds can be ionized and detected by any suitable means. A "mass spectrometer" typically includes an ionizer and an ion detector. Generally, one or more target molecules are ionized, and the ions are subsequently introduced into a mass spectrometry instrument, where, due to a combination of magnetic and electric fields, the ions follow a spatial path that depends on their mass ("m") and charge ("z"). The term "ionization" or "ionize" refers to the process of generating analyte ions with a net charge equal to one or more electron units. Negative ions are those with a net negative charge of one or more electron units, while positive ions are those with a net positive charge of one or more electron units. MS methods can be performed in a "negative ion mode" in which negative ions are generated and detected, or in a "positive ion mode" in which positive ions are generated and detected.

[0027] "Tandem mass spectrometry" or "MS / MS" involves multiple mass spectrometry selection steps, where analyte fragmentation occurs between stages. In a tandem mass spectrometer, ions are formed in an ion source and separated by their mass-to-charge ratio in the first stage of the mass spectrometry (MS1). Ions with a specific mass-to-charge ratio (precursor ions or parent ions) are selected, and fragment ions (or daughter ions) are formed by collision-induced dissociation, ion-molecule reactions, or photodissociation. The resulting ions are then separated and detected in the second stage of the mass spectrometry (MS2).

[0028] Although ionization sources such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are known, a reliable tool for ionization is laser desorption ionization (LDI), and a common laser for it is an ultraviolet (355 nm) Nd:YAG laser (neodymium-doped yttrium aluminum garnet; Nd:Y3Al5O 12 )

[0029] Since a mass spectrometer separates and detects ions with slightly different masses, it is easy to distinguish different isotopes of a given element. Therefore, mass spectrometry is an important method for the 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 global measurement of proteins in proteomics. Generally, de novo sequencing of peptides or proteins by mass spectrometry can be performed without prior knowledge of the amino acid sequence.

[0030] Mass spectrometry can be coupled with additional analytical methods, including chromatographic methods such as gas chromatography (GC), liquid chromatography (LC) especially HPLC, and / or ion mobility-based separation techniques. In the context of the present disclosure, the terms "analyte", "analyte molecule", or "target analyte" are used interchangeably and refer to a chemical substance to be analyzed via mass spectrometry. Chemical substances suitable for analysis via mass spectrometry, i.e., analytes, can be any kind of molecule present in a living organism, including but not limited to nucleic acids (e.g., DNA, mRNA, miRNA, rRNA, etc.), amino acids, peptides, proteins (e.g., cell surface receptors, cytoplasmic proteins, etc.), metabolites or hormones (e.g., testosterone, estrogen, estradiol, etc.), fatty acids, lipids, carbohydrates, steroids, ketosteroids, seco-steroids (e.g., vitamin D), molecules characterized by a certain modification of another molecule (e.g., sugar moiety or phosphoryl residue on a protein, methyl-residue on genomic DNA), substances internalized by an organism (e.g., therapeutic drugs, abused drugs, toxins, etc.) or metabolites of such substances. Such analytes can be used as biomarkers. In the context of the present invention, the term "biomarker" refers to a substance within a biological system that serves as an indicator of the biological state of said system. Most sample workflows in MS further include sample preparation and / or enrichment steps, where one or more target analytes are separated from the matrix using, for example, gas chromatography or liquid chromatography.

[0031] In some preferred embodiments of the derivatizing agent, the absorption maximum of chromophore C is the absorption maximum determined by UV / VIS spectroscopy. Preferably, the absorption maximum of chromophore C is in the range of 290 nm to 380 nm, more preferably in the range of 300 nm to 360 nm, and even more preferably in the range of 305 nm to 330 nm.

[0032] In some preferred embodiments of the derivatizing agent, chromophore C has the structure of formula (C)

[0033] where R 1 、R 2 、R 3 、R 4 、R 5 are independently selected from the group consisting of: a hydrogen atom; a hydroxy group; an NR x R y group, where R x and R y are independently a hydrogen atom or a C1 to C5 alkyl group; a C1 to C5 alkyl, a C5 to C10 (hetero)aryl, and an –O-C1 to C3 alkoxy group; and R 6is absent (i.e., there is a direct covalent single bond between the aromatic ring and the C(=O) group) or is a –CR 7 =CR 8 - group, where R 7 is a hydrogen atom or a C1 - C3 alkyl group and R 8 is selected from the group consisting of a hydrogen atom, a C1 - C5 alkyl group, and an electron - withdrawing group; and the dashed line represents a bond to the linker, and the bond is preferably a single bond.

[0034] In some preferred embodiments of the derivatizing agent of formula (C), R 1 , R 2 , R 3 , R 4 , R 5 are independently selected from the group: a hydrogen atom, a hydroxyl group, an NR x R y group, where R x and R y are independently a hydrogen atom or a C1 - C5 alkyl group and a –O - C1 - C3 alkoxy group. More preferably, R 1 , R 2 , R 3 , R 4 , R 5 are independently selected from the group: a hydrogen atom, a hydroxyl group, an NR x R y group, where R x and R y are independently a hydrogen atom or a C1 - C5 alkyl group and a –O - C1 - C3 alkoxy group, provided that at least one of R 1 , R 2 , R 3 , R 4 , R 5 is a hydroxyl group or a –O - C1 - C3 alkoxy group. The electron - withdrawing group of R 8 is preferably selected from the group consisting of a cyano group, a nitro group, a carboxyl group, a halogen atom (preferably a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom), and an aryl group, where "aryl" is preferably selected from the group: a C5 - C10 (hetero)aryl group, more preferably a phenyl group, and the electron - withdrawing group of R 8 is more preferably a cyano (C≡N) group.

[0035] In some preferred embodiments of the derivatizing agent, the chromophore C has a structure of formula (C1), (C2), or (C3):

[0036]

[0037] where R 1 , R 2 , R3 and R 4 are independently selected from the group consisting of: a hydrogen atom, a hydroxy group, NR x R y group, wherein R x and R y are independently a hydrogen atom or a C1-C5 alkyl group, C1-C5 alkyl, C5-C10 (hetero)aryl, and -O-C1-C3 alkoxy group.

[0038] Similarly for (C1), (C2), and (C3), which applies in some preferred embodiments where R 1 and R 2 and R 3 and R 4 and R 5 are independently selected from the group consisting of: a hydrogen atom, a hydroxy group, NR x R y group, wherein R x and R y are independently a hydrogen atom or a C1-C5 alkyl group and -O-C1-C3 alkoxy group. More preferably, R 1 and R 2 and R 3 and R 4 and R 5 are independently selected from the group consisting of: a hydrogen atom, a hydroxy group, NR x R y group, wherein R x and R y are independently a hydrogen atom or a C1-C5 alkyl group and -O-C1-C3 alkoxy group, provided that at least one of R 1 and R 2 and R 3 and R 4 and R 5 is a hydroxy group or -O-C1-C3 alkoxy group. The electron-withdrawing group of R 8 is preferably selected from the group consisting of a cyano group, a nitro group, a carboxyl group, a halogen atom (preferably a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom), and an aryl group, wherein "aryl" is preferably selected from the group consisting of: C5-C10 (hetero)aryl groups, more preferably a phenyl group, wherein the electron-withdrawing group of R 8 is more preferably a cyano (C≡N) group.

[0039] In some preferred embodiments of the derivatizing agent, the chromophore C has the structure of formula (C1), wherein R 1 and R 4 are both methoxy groups and R 3 is a hydroxy group (C1, wherein these residues R 2 and R 3 and R4 For the remainder of sinapic acid). In some preferred embodiments of the derivatizing agent, the chromophore C has the structure of formula (C2), where R 2 and R 4 are both hydroxyl groups (C2, where these residues R 2 、R 4 are the remainder of 2,5-dihydroxybenzoic acid). In some preferred embodiments of the derivatizing agent, the chromophore C has the structure of formula (C3), where R 3 is a hydroxyl group (C3, where the residue R 3 is the remainder of α-cyano-4-hydroxy-cinnamic acid).

[0040] In some preferred embodiments of the derivatizing agent, the linker L1 is selected from the group consisting of: (C1-C5 alkylene-O-) m group, where m is an integer in the range of 1 to 10, C1 to C20 alkylene groups, C1 to C20 alkylene-heteroaryl groups, and (C1-C5 alkylene)-O-(C1-C5 alkylene) groups, optionally linked or cross-linked to a unit selected from the group consisting of (hetero)aryl groups, N2, NO, NO2, S2, SO, SO2, CO, and CO2, and if present, the unit is preferably a heteroaryl group, more preferably from triazole, phenyltriazole, tetrazole, and phenyltetrazole. "Heteroaryl" is preferably a C1 to C10 heteroaryl having at least one heteroatom as part of the ring structure, where the at least one heteroatom is preferably selected from N, O, and S, more preferably the heteroaryl is selected from the group consisting of triazole, tetrazole, tetrazine, oxadiazole, thiadiazole, and any hydrogenated derivatives thereof, more preferably selected from the group consisting of 1,2,3-triazole, 1,2,4-triazole, 1,4,5-triazole, 3,4,5-triazole, 1,2,3,4-tetrazole, 2,3,4,5-tetrazole, 2,3,5,6-tetrazole, and 1,2,4,5-tetrazine. In some preferred embodiments of the derivatizing agent, the linker L1 has the structure (L1a) q -(L1b) r -(L1c) s , where q, r, s are each zero or 1, provided that at least one of q, r, s is 1; L1a is selected from (C1-C5 alkylene-O-) ma group, where m is an integer in the range of 1 to 10, a C1-C10 alkylene group; L1b is a unit selected from the group consisting of: N2, NO, NO2, S2, SO, SO2, CO, CO2, triazole, phenyltriazole, tetrazole, and phenyltetrazole, where the unit is preferably triazole or tetrazole, more preferably the unit N is triazole, and more preferably the unit N is a 1,2,3-triazole ring, which is bonded to the linker L1a (if present) via a single bond at position 1 (N atom) of the triazole ring and bonded to the linker L1c (if present) via a single bond at position 4 (C atom) of the triazole ring; L1c (if present) is selected from the group consisting of: (C1-C5 alkylene-O-) m a group, where m is an integer in the range of 1 to 10, a C1-C10 alkylene, and a (C1-C5 alkylene)-O-(C1-C5 alkylene) group. In some preferred embodiments of the derivatizing agent, the linker L1 has the structure

[0041]

[0042] where the dashed lines respectively represent the bonds to (C) and (Z).

[0043] In some preferred embodiments of the derivatizing agent, the charged unit Z is positively charged or negatively charged, preferably positively charged. In some preferred embodiments of the derivatizing agent, the charged unit Z is positively charged, and preferably is a tetraalkylammonium group, more preferably -CH2N + (CH3)2CH2-group. In some preferred alternative embodiments of the derivatizing agent, the charged unit Z is negatively charged, where the negatively charged unit Z is preferably selected from the group consisting of phosphate, sulfate, sulfonate, and carboxylate.

[0044] In some preferred embodiments of the derivatizing agent, the linker L2 contains 1 to 10 C atoms and optionally one or more heteroatoms. Preferably, the linker L2 is a C1-C5-alkylene-C5 to C10 aromatic ring, which preferably bears a C(=O) unit as a substituent of the aromatic ring. The linker L2 is more preferably a C1-C3-alkylene-C6 aromatic ring, which preferably bears a C(=O) unit as a substituent of the aromatic ring, where the C6 aromatic ring is more preferably a substituted or unsubstituted benzene ring, more preferably a benzene ring, which bears a C(=O) group at a position of the benzene ring, which is para to the position where it is bonded to the C1-C3-alkylene, and more preferably bears no other substituents.

[0045] In some preferred embodiments of the derivatizing agent, the reactive group X is selected from the group consisting of: a carbonyl-reactive unit, a diene-reactive unit, a hydroxy-reactive unit, an amino-reactive unit, an imine-reactive unit, a thiol-reactive unit, a diol-reactive unit, a phenol-reactive unit, an epoxide-reactive unit, a disulfide-reactive unit, and an azide-reactive unit.

[0046] In some preferred embodiments of the derivatizing agent, the reactive unit X is a carbonyl-reactive unit, which is capable of reacting with any type of molecule having a carbonyl group. The carbonyl-reactive unit is preferably selected from the group consisting of: a carboxy-reactive unit, a keto-reactive unit, an aldehyde-reactive unit, an anhydride-reactive unit, a carbonyl ester-reactive unit, and an imide-reactive unit. In some preferred embodiments of the derivatizing agent, the carbonyl-reactive unit may have a supernucleophilic N atom NH2-N / O strengthened by the a-effect of an adjacent O or N atom or have a dithiol molecule.

[0047] In some preferred embodiments of the derivatizing agent, the carbonyl-reactive unit is selected from the group consisting of:

[0048] (i) a hydrazine unit, for example, H2N-NH- or H2N-NR a - unit, where R a is aryl, aryl containing one or more heteroatoms, or C1-4 alkyl, especially C1 or C2 alkyl, which is optionally substituted, for example, by halogen, hydroxy, and / or C1-3 alkoxy,

[0049] (ii) a hydrazide unit, especially a carbohydrazide or sulfohydrazide unit, especially H2N-NH-C(O)- or H2N-NR b -C(O)- unit, where R b is aryl, aryl containing one or more heteroatoms, or C1-4 alkyl, especially C1 or C2 alkyl, which is optionally substituted, for example, by halogen, hydroxy, and / or C1-3 alkoxy,

[0050] (iii) a hydroxylamino unit, for example, the H2N-O- unit, and

[0051] (iv) a dithiol unit, especially a 1,2-dithiol or 1,3-dithiol unit.

[0052] In some preferred embodiments of the derivatizing agent, where the carbonyl-reactive unit is a carboxy-reactive unit, the carboxy-reactive unit reacts with the carboxy group on the analyte molecule. In an embodiment of the first aspect of the present invention, the carboxy-reactive unit is selected from the group consisting of: a diazo unit, an alkyl halide, an amine, and a hydrazine unit.

[0053] In some preferred embodiments of the derivatizing agent, the reactive unit X is a diene-reactive unit capable of reacting with an analyte containing a diene group. In some preferred embodiments of the derivatizing agent, the diene-reactive units are selected from the group consisting of Cookson-type reagents, such as 1,2,4-triazoline-3,5-dione, which can act as a dienophile.

[0054] In some preferred embodiments of the derivatizing agent, the reactive unit X is a hydroxy-reactive unit capable of reacting with an analyte containing a hydroxy group. In some preferred embodiments of the derivatizing agent, the hydroxy-reactive units are selected from the group consisting of sulfonyl chlorides, activated carboxylic acid esters (NHS, or imidazole anions), and fluorinated arenes / heteroarenes capable of nucleophilic substitution of fluorine (T. Higashi J Steroid Biochem Mol Biol. September 2016; l62:57-69). In some preferred embodiments of the derivatizing agent, the reactive unit X is a diol-reactive unit that reacts with a diol group on the analyte molecule. In some preferred embodiments of the derivatizing agent, where the reactive unit is a 1,2-diol-reactive unit, the 1,2-diol-reactive unit contains boric acid. In a further embodiment, the diol can be oxidized to the corresponding ketone or aldehyde and then react with the ketone / aldehyde-reactive unit X. In some preferred embodiments of the derivatizing agent, the amino-reactive unit reacts with an amino group on the analyte molecule. In some preferred embodiments of the derivatizing agent, the amino-reactive units are selected from the group consisting of active ester groups such as N-hydroxysuccinimide (NHS) esters or sulfo-NHS esters, pentafluorophenyl esters, carbonyl imidazole esters, squaric acid esters, hydroxybenzotriazole (HOBt) esters, 1-hydroxy-7-azabenzotriazole (HOAt) esters, and sulfonyl chloride units.

[0055] In some preferred embodiments of the derivatizing agent, the thiol-reactive unit reacts with a thiol group on the analyte molecule. In some preferred embodiments of the derivatizing agent, the thiol-reactive units are selected from the group consisting of haloacetyl groups, particularly selected from the group consisting of Br / I-CH2-C(=O)- units, acrylamide / acrylic acid ester units, unsaturated imide units such as maleimide, mesylphenyl oxadiazole, and sulfonyl chloride units.

[0056] In some preferred embodiments of the derivatizing agent, the phenolic reactive unit reacts with the phenolic group on the analyte molecule. In some preferred embodiments of the derivatizing agent, the phenolic reactive unit is selected from the group consisting of: active ester groups such as N-hydroxysuccinimide (NHS) ester or sulfo-NHS ester, pentafluorophenyl ester, carbonyl imidazole ester, squaric acid ester, hydroxybenzotriazole (HOBt) ester, 1-hydroxy-7-azabenzotriazole (HOAt) ester, and sulfonyl chloride unit. The phenolic group present on the analyte molecule can react with triazodione via reaction (H. Ban et al. J. Am. Chem. Soc., 2010, 132(5), pp. 1523-1525), or alternatively by diazotization or by ortho-nitration followed by reduction to an amine, and then the amine reacts with an amine-reactive reagent.

[0057] In some preferred embodiments of the derivatizing agent, the reactive unit X is an epoxide-reactive unit that is capable of reacting with an analyte containing an epoxide group. In some preferred embodiments of the derivatizing agent, the epoxide-reactive unit is selected from the group consisting of: amino, thiol, a supernucleophilic N atom NH2-N / O molecule enhanced by the a-effect of adjacent O or N atoms.

[0058] In some preferred embodiments of the derivatizing agent, the epoxide-reactive unit is selected from the following group:

[0059] (i) hydrazine units, for example, H2N-NH- or H2N-NR a - unit, where R 1 is aryl, aryl containing one or more heteroatoms, or C1-4 alkyl, especially C1 or C2 alkyl, which is optionally substituted, for example, by halogen, hydroxy, and / or C1-3 alkoxy,

[0060] (ii) acylhydrazine units, especially carbohydrazide or sulfohydrazide units, especially H2N-NH-C(O)- or H2N-NR b -C(O)- unit,

[0061] where R b is aryl, aryl containing one or more heteroatoms, or C1-4 alkyl, especially C1 or C2 alkyl, which is optionally substituted, for example, by halogen, hydroxy, and / or C1-3 alkoxy, and

[0062] (iii) hydroxyamino units, for example, H2N-O- unit.

[0063] In some preferred embodiments of the derivatizing agent, the reactive unit X is a disulfide-reactive unit, which is capable of reacting with an analyte containing a disulfide group. In some preferred embodiments of the derivatizing agent, the disulfide-reactive unit is selected from the group consisting of thiols. In other embodiments, the disulfide group can be reduced to the corresponding thiol group and then reacted with the thiol-reactive unit X.

[0064] In some preferred embodiments of the derivatizing agent, the reactive unit X is an azide-reactive unit, which reacts with an azide group on an analyte molecule. In some preferred embodiments of the derivatizing agent, the azide-reactive unit reacts with the azide group by azide-alkyne cycloaddition. In some preferred embodiments of the derivatizing agent, the azide-reactive unit is selected from the group consisting of: alkynes (alkyl or aryl), linear alkynes or cyclic alkynes. The reaction between the azide and the alkyne can be carried out with or without a catalyst. In other embodiments of the first aspect of the present invention, the azide group can be reduced to the corresponding amino group and then reacted with the amino-reactive unit X.

[0065] Aspect 2 - kit

[0066] A second aspect of the present invention relates to a kit comprising a derivatizing agent according to the first aspect. All details, embodiments and preferred embodiments disclosed above in the part related to the first aspect also apply to the kit of the second aspect.

[0067] A "kit" is any article (e.g., a package or container) containing at least one reagent of the present invention, which reagent is, for example, a medicine for treating a disease or a probe for specifically detecting a biomarker gene or protein. The kit is preferably promoted, distributed or sold as a unit for carrying out the uses and / or methods of the present invention, as described in the parts related to the third and fourth aspects of the present invention below.

[0068] Generally, the kit may further comprise a carrier mechanism that partitions compartments to receive one or more container mechanisms (such as vials, tubes, etc.) within a strictly defined space. In particular, each of the container mechanisms includes one of the independent elements to be used in the method of the first aspect. The kit may further comprise one or more other containers containing other materials, which other materials include but are not limited to buffers, diluents, filters, needles, syringes and package inserts with instructions for use. Labels may be present on the containers to indicate the use of the composition for a specific application and may also indicate guidelines for in vivo or in vitro use. Computer program code may be provided on a data storage medium or device such as an optical storage medium (e.g., a compact disc) or directly on a computer or data processing device. In addition, the kit may contain a standard amount of a biomarker for calibration purposes as described elsewhere herein.

[0069] "Package insert" is used to refer to the instructions that are usually included in the commercial packaging of a therapeutic product or a medicine, which contain information on indications, usage, dosage, administration, contraindications, other therapeutic products to be used in combination with the packaged product, and / or warnings regarding the use of such therapeutic product or medicine.

[0070] Aspect 3 - Use of a derivatizing agent for mass spectrometry of an analyte molecule

[0071] In a third aspect, the present invention relates to the use of a derivatizing agent according to the first aspect for the mass spectrometry of an analyte molecule, wherein the mass spectrometry is laser desorption ionization mass spectrometry (LDI-MS), preferably (MA)LDI-MS, more preferably (MA)LDI-MS / MS, or (SA)LDI-MS, more preferably (SA)LDI-MS / MS. All the details, examples, and preferred embodiments disclosed above in the parts related to the first and second aspects are also applicable to the use in the third aspect. "MS" and "MS / MS" have the meanings explained in the above part related to the first aspect.

[0072] The term "LDI" is a common abbreviation for laser desorption ionization, which is called matrix-assisted laser desorption ionization "MALDI" in applications in combination with a suitable matrix. Matrix materials and supports, such as metals, especially steel plates, are known to those skilled in the art and are equally applicable to the applicable conditions. The matrix usually consists of crystalline molecules, and the three most commonly used ones are sinapic acid, α-cyano-4-hydroxycinnamic acid (α-CHCA, α-cyano or α-matrix), and 2,5-dihydroxybenzoic acid (DHB). The MALDI technique generally involves the use of UV lasers, such as nitrogen lasers (337 nm) and frequency-tripled and frequency-quadrupled Nd:YAG lasers (355 nm and 266 nm, respectively). The infrared laser wavelengths for infrared MALDI include 2.94 μm Er:YAG lasers, mid-IR optical parametric oscillators, and 10.6 μm carbon dioxide lasers.

[0073] Surface-assisted laser desorption ionization "SALDI" is a soft laser desorption technique that does not use matrix molecules, but instead uses a medium that absorbs energy from the laser and then transfers the energy to the target sample, where the active surface of a specific matrix plays a decisive role. An important substrate is the solid surface of porous silicon. Porous silicon represents the first matrix-free SALDI surface analysis, which allows for the easy detection of intact molecular ions. It is well known that a variety of different surfaces of SA can be used as SALDI substrates. Depending on the component composition, most SALDI substrates reported in the literature can generally be classified into three main types: carbon-based, semiconductor-based, and metal-based. The SALDI process using inorganic matrices for preparation has been described in several works, for example, Law et al., (Anal. Bioanal. Chem. 2011, 399, 2597, DOI 10.1007 / s00216-010-4063-3). Preferably, in the context of the present invention, a SALDI-MS target plate with a functional amorphous a-C:H:Si:X (X = heteroatom modification) plasma-activated chemical vapor deposition (PACVD) surface coating based on a steel plate is used as the top layer.

[0074] Laser desorption ionization mass spectrometry (LDI-MS) measurements are preferably carried out in the positive ion mode using a Nd:YAG laser wavelength of 355 nm. The laser repetition rate of the LDI-MS system is set respectively to be suitable for MALDI experiments and SALDI experiments. Other parameters of the measurement, such as the moving mode, moving speed, frequency, acquisition time, mass spectrometry scan time, laser intensity, or voltage setting, are appropriately selected, and these parameters are known to those skilled in the art who are familiar with the required software-based data analysis tools.

[0075] Aspect 4 - conjugate

[0076] The fourth aspect of the present invention relates to a conjugate of a derivatizing agent and an analyte according to the first aspect of the present invention, wherein the conjugate has a structure of formula (II),

[0077] C–L1–Z–(L2) p –Xa–Ya-A (II)

[0078] wherein C, L1, L2, p, Z and N are as defined in the portion related to the first aspect; Xa is the remainder of the reactive group X as defined in the portion related to the first aspect; A is an analyte and Ya is the remainder of the reactive group Y bound to the analyte A, which has reacted with the reactive group X of the derivatizing agent, thereby forming a covalent bond between Xa and Ya. All details, examples and preferred embodiments described above in the portions related to the first, second and third aspects of the present invention also apply to the conjugates of the fourth aspect, in particular all details, examples and preferred embodiments described above in the portion related to the first aspect also apply herein.

[0079] In some preferred embodiments of the conjugate, the analyte is selected from the group consisting of: nucleic acids (preferably selected from DNA, mRNA, miRNA and rRNA), amino acids, peptides, proteins (preferably cell surface receptors or cytosolic proteins), metabolites, hormones (preferably selected from testosterone, estrogen and estradiol), fatty acids, lipids, carbohydrates, steroids, ketosteroids, secosteroids (vitamin D), certain modified molecular features of another molecule (preferably selected from sugar moieties, phosphoryl residues on proteins, methyl residues on genomic DNA), substances internalized in a living organism (preferably selected from therapeutic drugs, abused drugs, toxins) and metabolites of such substances.

[0080] The analyte may be present in a target sample (such as a biological sample and a clinical sample). The terms "sample" or "target sample" are used interchangeably herein and refer to a part or section of a tissue, organ or individual, usually smaller than such tissue, organ or individual, and is intended to represent the whole tissue, organ or individual. When analyzed, the sample provides information about the state of the tissue or the health or diseased state of the organ or individual. Examples of samples include, but are not limited to: liquid samples such as blood, serum, plasma, synovial fluid, cerebrospinal fluid, urine, saliva and lymph fluid; or solid samples such as dried blood spots and tissue extracts. Other examples of samples are cell cultures or tissue cultures.

[0081] In the context of the present disclosure, a sample may be derived from an "individual" or a "subject". Generally, the subject is a mammal. Mammals include, but are not limited to, domesticated animals (such as cows, sheep, cats, dogs and horses), primates (such as humans and non-human primates, such as monkeys), rabbits and rodents (such as mice and rats).

[0082] Before analysis via mass spectrometry, the sample can be pre-treated in a manner specific to the particular sample and / or analyte. In the context of the present disclosure, the term "pre-treatment" refers to any measures necessary to allow subsequent analysis of the desired analyte via mass spectrometry. Pre-treatment measures typically include, but are not limited to, eluting solid samples (e.g., eluting dried blood spots), adding a hemolysis reagent (HR) to a whole blood sample, and adding an enzyme reagent to a urine sample. The addition of an internal standard (ISTD) is also considered pre-treatment of the sample.

[0083] The term "hemolysis reagent (HR)" refers to a reagent that lyses the cells present in the sample. In the context of the present invention, the hemolysis reagent particularly refers to a reagent that lyses the cells present in a blood sample (including, but not limited to, red blood cells present in a whole blood sample). A well-known hemolysis reagent is water (H2O). Other examples of hemolysis reagents include, but are not limited to, deionized water, hypertonic liquids (e.g., 8M urea), ionic liquids, and different detergents.

[0084] Typically, an internal standard (ISTD) is a known amount of a substance that exhibits characteristics similar to those of the target analyte when subjected to a mass spectrometry detection workflow (i.e., including any pre-treatment, enrichment, and actual detection steps). Although the ISTD exhibits characteristics similar to those of the target analyte, it can still be clearly distinguished from the target analyte. For example, during chromatographic separation such as gas chromatography and liquid chromatography, the ISTD has a retention time approximately the same as that of the target analyte from the sample. Thus, both the analyte and the ISTD enter the mass spectrometer simultaneously. However, the ISTD exhibits a different molecular mass from the target analyte from the sample. This enables mass spectrometry discrimination between the ions from the ISTD and the ions from the analyte by their different mass-to-charge (m / z) ratios. Both undergo fragmentation and provide daughter ions. These daughter ions can be distinguished from each other and from their respective parent ions by their m / z ratios. Thus, the signals from the ISTD and the analyte can be determined and quantified independently. Since the amount of the ISTD added is known, the signal intensity of the analyte from the sample can be attributed to a specific quantitative amount of the analyte. Thus, the addition of the ISTD allows for a relative comparison of the amounts of the detected analyte and enables unambiguous identification and quantification of the analyte when the target analyte present in the sample reaches the mass spectrometer. Typically but not necessarily, the ISTD is an isotopically labeled variant of the target analyte (containing labels such as 2H, 13C, or 15N, etc.).

[0085] In addition to pretreatment, the sample may undergo one or more enrichment steps. In the context of the present disclosure, the term "first enrichment process" or "first enrichment workflow" refers to an enrichment process that occurs after pretreatment of the sample and provides a sample containing an analyte enriched relative to the initial sample. The first enrichment workflow may include chemical precipitation (e.g., using acetonitrile) or use of a solid phase. Suitable solid phases include, but are not limited to, solid phase extraction (SPE) cartridges and beads. The beads can be non-magnetic, magnetic, or paramagnetic. The beads can be differentially coated to have specificity for the target analyte. Depending on the intended use, i.e., depending on the intended capture molecule, the coating can vary. Which coating is suitable for which analyte is well known to those skilled in the art. The beads can be made of a variety of different materials. The beads can have a variety of sizes and contain a surface with or without pores.

[0086] In the context of the present disclosure, the term "second enrichment process" or "second enrichment workflow" refers to an enrichment process that occurs after pretreatment of the sample and the first enrichment process and provides a sample containing an analyte enriched relative to the initial sample and the sample after the first enrichment process.

[0087] In some preferred embodiments of the conjugate, the reactive group Y is selected from the group consisting of: a carbonyl group, a diene group, a hydroxyl group, an amine group, an imine group, a thiol group, a diol group, a phenol group, an epoxide group, a disulfide group, and an azide group.

[0088] In some preferred embodiments of the conjugate, the analyte molecule contains a functional group selected from the above group prior to reacting with the derivatizing agent, wherein each functional group indicated in the group is capable of forming a covalent bond with the reactive unit X of the derivatizing agent. Additionally, it is contemplated within the scope of the present invention that functional groups present on the analyte molecule will first be converted into another group that is more readily reactive with the reactive unit X of the derivatizing agent.

[0089] In some embodiments, the analyte molecule contains a carbonyl group as a functional group prior to reacting with the derivatizing agent, the carbonyl group being selected from the group consisting of: a carboxylic acid group, an aldehyde group, a ketone group, a masked aldehyde, a masked ketone group, an ester group, an amide group, and an anhydride group. In embodiments where the carbonyl group is an amide group, it is well known to those skilled in the art that such an amide group is a stable group, but it can be hydrolyzed to convert the amide group into a formic acid group and an amino group. The hydrolysis of the amide group can be achieved via an acid / base catalyzed reaction or an enzymatic process, either of which is well known to those skilled in the art. In embodiments where the carbonyl group is a masked aldehyde group or a masked ketone group, the corresponding group is a hemiacetal group or an acetal group, particularly a cyclic hemiacetal group or an acetal group. In some embodiments, the acetal group is converted into an aldehyde or ketone group prior to reacting with the derivatizing agent.

[0090] In some embodiments, the carbonyl group is a ketone group. The ketone group can be transferred to an intermediate imine group prior to reacting with the reactive unit of the derivatizing agent. In some embodiments, the analyte molecule comprising one or more ketone groups is preferably a ketosteroid. In certain embodiments, the ketosteroids are selected from the group consisting of: testosterone, epitestosterone, dihydrotestosterone (DHT), desoxymethyltestosterone (DMT), tetrahydrogestrinone (THG), aldosterone, estrone, 4-hydroxyestrone, 2-methoxyestrone, 2-hydroxyestrone, 16-ketoestradiol, 16α-hydroxyestrone, 2-hydroxyestrone-3-methyl ether, prednisone, prednisolone, pregnenolone, progesterone, DHEA (dehydroepiandrosterone), 17-OH pregnenolone, 17-OH progesterone, 17-OH progesterone, androsterone, epiandrosterone, and δ4-androstenedione) 11-deoxycortisol corticosterone, 21-deoxycortisol, 11-deoxycorticosterone, allopregnanolone, and aldosterone.

[0091] In some embodiments, the carbonyl group is a carboxyl group. The carboxyl group reacts directly with the derivatizing agent or is converted to an activated ester group prior to reacting with the derivatizing agent. In some embodiments, the analyte molecule comprising one or more carboxyl groups is selected from the group consisting of: D8-tetrahydrocannabinolic acid, benzoylecgonine, salicylic acid, 2-hydroxybenzoic acid, gabapentin, pregabalin, valproic acid, vancomycin, methotrexate, mycophenolic acid, montelukast, repaglinide, furosemide, telmisartan, gemfibrozil, diclofenac, ibuprofen, indomethacin, zomepirac, isoxepac, and penicillin. In some embodiments, the analyte molecule comprising one or more carboxyl groups is preferably an amino acid preferably selected from the group consisting of: arginine, lysine, aspartic acid, glutamic acid, glutamine, asparagine, histidine, serine, threonine, tyrosine, cysteine, tryptophan, alanine, isoleucine, leucine, methionine, phenylalanine, valine, proline, and glycine.

[0092] In some embodiments, the carbonyl group is an aldehyde group. The aldehyde group can be transferred to an intermediate imine group prior to reacting with the reactive unit of the derivatizing agent. In some embodiments, the analyte molecule comprising one or more aldehyde groups is preferably selected from the group consisting of: pyridoxal, N-acetyl-D-glucosamine, acrivastine, streptomycin, josamycin.

[0093] In some embodiments, the carbonyl group is a carbonyl ester group. The analyte molecule comprising one or more ester groups is preferably selected from the group consisting of: cocaine, heroin, methylphenidate, aceclofenac, acetylcholine, amcinonide, amiloride, amethocaine, ampicloxacillin, aripiprazole, and artesunate, pethidine.

[0094] In some embodiments, the carbonyl group is an anhydride group. In some embodiments, the analyte molecule comprising one or more anhydride groups is preferably selected from the group consisting of cantharidin, succinic anhydride, trimellitic anhydride, and maleic anhydride.

[0095] In some embodiments, the analyte molecule comprises one or more diene groups, particularly conjugated diene groups, as functional groups. The analyte molecule comprising one or more diene groups is preferably an open-ring steroid. In some embodiments, the open-ring steroids are selected from the group consisting of cholecalciferol (vitamin D3), ergocalciferol (vitamin D2), calcifediol, calcitriol, tachysterol, lumisterol, and tacalcitol. In particular, the open-ring steroid is vitamin D, particularly vitamin D2 or D3 or derivatives thereof. In certain embodiments, the open-ring steroids are selected from the group consisting of vitamin D2, vitamin D3, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 3-epi-25-hydroxyvitamin D2, 3-epi-25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D2, 1,25-dihydroxyvitamin D3, 24,25-dihydroxyvitamin D2 and 24,25-dihydroxyvitamin D3, vitamin A, tretinoin, isotretinoin, alitretinoin, natamycin, sirolimus, amphotericin B, nystatin, everolimus, temsirolimus, fidaxomicin.

[0096] In some embodiments, the analyte molecule comprises one or more hydroxyl groups as functional groups. Then, the analyte molecule preferably comprises a single hydroxyl group or two hydroxyl groups. In embodiments where there are more than one hydroxyl group, the two hydroxyl groups can be positioned adjacent to each other (1,2-diol), or can be separated by 1, 2, or 3 C atoms (1,3-diol, 1,4-diol, 1,5-diol, respectively). In certain embodiments, the analyte molecule comprises a 1,2 diol group. In embodiments where only one hydroxyl group is present, the analyte is preferably selected from the group consisting of primary alcohols, secondary alcohols, and tertiary alcohols. In some embodiments where the analyte molecule comprises one or more hydroxyl groups, the analyte is preferably selected from the group consisting of benzyl alcohol, menthol, L-carnitine, pyridoxine, metronidazole, isosorbide mononitrate, guaifenesin, clavulanate, Migitol, zalcitabine, isoproterenol, acyclovir, methocarbamol, tramadol, venlafaxine, atropine, clofedanol, α-hydroxyalprazolam, α-hydroxytriazolam, lorazepam, nordazepam, temazepam, ethyl glucuronide, ethylmorphine, morphine, morphine-3-glucuronide, buprenorphine, codeine, dihydrocodeine, p-hydroxypropoxyphene, O-demethyltramadol, dihydroquinidine, and quinidine. In some embodiments where the analyte molecule comprises more than one hydroxyl group, the analyte is preferably selected from the group consisting of vitamin C, glucosamine, mannitol, tetrahydrobiopterin, cytarabine, azacitidine, ribavirin, floxuridine, gemcitabine, streptozotocin, adenosine, vidarabine, cladribine, estriol, trifluridine, clofarabine, nadolol, zanamivir, lactulose, adenosine monophosphate, idoxuridine, Regadenoson, lincomycin, clindamycin, Canaglifozin, tobramycin, netilmicin, kanamycin, ticagrelor, epirubicin, doxorubicin, arbekacin, streptomycin, Quabain, amikacin, neomycin, Framycetin, paromomycin, erythromycin, clarithromycin, azithromycin, vindesine, digitoxin, digoxin, metrizamide, acetyl digitoxin, deslanoside, fludarabine, clofarabine, gemcitabine, cytarabine, capecitabine, vidarabine, trifluridine, idoxuridine, and plicamycin.

[0097] In some embodiments, the analyte molecule comprises one or more thiol groups (including but not limited to alkyl - thiol and thiol - aryl groups) as functional groups. Analyte molecules comprising one or more thiol groups are preferably selected from the group consisting of: thiomandelic acid, DL - captopril, DL - thiorphan, N - acetylcysteine, D - penicillamine, glutathione, L - cysteine, zofenoprilat, tiopronin, dimercaprol, and succimer.

[0098] In some embodiments, the analyte molecule comprises one or more disulfide groups as functional groups. Analyte molecules comprising one or more disulfide groups are preferably selected from the group consisting of: glutathione disulfide, dithiooxypyridine, selenium disulfide, disulfiram, lipoic acid, L - cystine, fursultiamine, octreotide, desmopressin, vapreotide, terlipressin, linaclotide, peginesatide.

[0099] In some embodiments, the analyte molecule comprises one or more epoxide groups as functional groups. Analyte molecules comprising one or more epoxide groups are preferably selected from the group consisting of: carbamazepine 10,11 - epoxide, carfilzomib, furosemide epoxide, fosfomycin, sevelamer, cerulenin, scopolamine, tiotropium bromide, methscopolamine bromide, eplerenone, mupirocin, natamycin, carfilzomib, oleandomycin.

[0100] In some embodiments, the analyte molecule comprises one or more phenol groups as functional groups. Analyte molecules comprising one or more phenol groups are preferably steroids or steroid - like compounds. In some embodiments, analyte molecules comprising one or more phenol groups preferably have sp 2Steroid or steroid-like compounds having a hybridized A ring and an OH group at the 3-position of the A ring. The steroid or steroid-like analyte molecule is preferably selected from the group consisting of: estrogens, estrogen-like compounds, estrone (E1), estradiol (E2), 17α-estradiol, 17β-estradiol, estriol (E3), 16-epiestriol, 17-epiestriol, and 16,17-epiestriol and / or their metabolites. In an embodiment, the metabolites are selected from the group consisting of: estriol, 16-epiestriol (16-epiE3), 17-epiestriol (17-epiE3), 16,17-epiestriol (16,17-epiE3), 16-ketoestradiol (16-ketoE2), 16α-hydroxyestrone (16α-OHE1), 2-methoxyestrone (2-MeOE1), 4-methoxyestrone (4-MeOE1), 2-hydroxyestrone-3-methyl ether (3-MeOE1), 2-methoxyestradiol (2-MeOE2), 4-methoxyestradiol (4-MeOE2), 2-hydroxyestrone (2OHE1), 4-hydroxyestrone (4-OHE1), 2-hydroxyestradiol (2-OHE2), estrone (E1), estrone sulfate (E1s), 17α-estradiol (E2α), 17β-estradiol (E2β), estradiol sulfate (E2s), equilin (Eq), 17α-dihydroequilin (Eqα), 17β-dihydroequilin (Eqβ), equilenin (EN), 17-dihydroequilenin (ENα), 17β-dihydroequilenin (ENβ), Δ8,9-dehydroestrone (dE1), Δ8,9-dehydroestrone sulfate (dE1s), Δ9-tetrahydrocannabinol, mycophenolic acid.

[0101] In some embodiments, the analyte molecule contains an amine group as a functional group. The amine group is preferably an alkyl-amine or aryl-amine group. In some embodiments, the analyte containing one or more amine groups is selected from the group consisting of: proteins and peptides. The analyte molecule containing an amine group is preferably selected from the group consisting of: 3,4-methylenedioxyamphetamine, 3,4-methylenedioxy-N-ethylamphetamine, 3,4-methylenedioxymethamphetamine, amphetamine, methamphetamine, N-methyl-1,3-benzodioxolylbutanamine, 7-aminonitrazepam, 7-aminoflunitrazepam, 3,4-dimethylmethcathinone, 3-fluoromethcathinone, 4-methoxymethcathinone, 4-methylethcathinone, 4-methylmethcathinone, amfepramone, 2-methylamino-1-(3',4'-methylenedioxyphenyl)butan-1-one (Butylone), ethcathinone, flephedrone, methcathinone, methylenedioxymethcathinone (Methylone), methylenedioxypyrovalerone, benzoylecgonine, deshydrodesmethylketamine, ketamine, norketamine, methadone, normethadone, 6-acetylmorphine, diacetylmorphine, morphine, norhydrocodone, oxycodone, oxymorphone, phencyclidine, norpropoxyphene, amitriptyline, clomipramine, dosulepin, doxepin, imipramine, nortriptyline, trimipramine, fentanyl, glycylxylidide, lidocaine, monoethylglycylxylidide, N-acetylprocainamide, procainamide, pregabalin, 2-methylamino-1-(3,4-methylenedioxyphenyl)butane, 2-amino-1-(3,4-methylenedioxyphenyl)butane, normeperidine, O-demethyltramadol, tramadol, lidocaine, N-acetylprocainamide, procainamide, gabapentin, lamotrigine, theophylline, amikacin, gentamicin, tobramycin, vancomycin, methotrexate, gabapentin, sisomicin, and 5-methylcytosine.

[0102] In some embodiments, the analyte molecule is a carbohydrate or a substance having a carbohydrate moiety, such as a glycoprotein or a nucleoside. Then the analyte molecule is preferably a monosaccharide, particularly selected from the group consisting of: ribose, deoxyribose, arabinose, ribulose, glucose, mannose, galactose, fucose, fructose, N-acetylglucosamine, N-acetylgalactosamine, neuraminic acid, N-acetylneuraminic acid, etc. In some embodiments, the analyte molecule is an oligosaccharide, particularly selected from the group consisting of: disaccharides, trisaccharides, tetrasaccharides, polysaccharides. In some embodiments, the disaccharides are preferably selected from the group consisting of: sucrose, maltose, and lactose. In some embodiments, the analyte molecule is a substance containing the above monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide moiety.

[0103] In some embodiments, the analyte molecule comprises an azide group as a functional group, which is selected from the group consisting of: alkyl azide or aryl azide. Then, the analyte molecule comprising one or more azide groups is preferably selected from the group consisting of zidovudine and azidocillin.

[0104] The functional group Y of the analyte reacts with or is capable of reacting with the reactive group X of the derivatizing agent; the reactive group X of the derivatizing agent is disclosed in detail in the part related to the first aspect of the present invention, where the suitable bonding with the functional group Y of the analyte is also described. Those skilled in the art should understand what the remaining parts Xa and Ya of X and Y are respectively: in each case, a covalent bond is formed. For example, if the reactive group X of the derivatizing agent is a carbonyl-reactive unit, such as a hydrazine unit, especially the a–NH-NH2 group, and the functional group of the analyte is a carbonyl group, such as a ketone group C(=O), then an –NH-N= structure is formed, and the remaining part Xa of X is –NH-N= and the remaining part Ya of Y is a carbon atom with a double bond to the nitrogen atom.

[0105] As already pointed out above, the chromophore C carried by the derivatizing agent is a suitable chromophore capable of achieving efficient energy transfer during LDI. In addition, the derivatizing agent adds a sufficiently large molecular weight to the target analyte, where the weight addition causes a sufficient mass shift beyond the high-noise background in the low molecular weight region of the biological sample. Generally, such a high molecular weight is known to result in adverse ionization characteristics (poor ionization efficiency, multiple fragmentation processes...), however, in this instance, this is circumvented by the presence of a permanent positive charge (the Z unit in formula (I)). Thus, the precursor ions (parent ions) can be detected with high sensitivity, and the precursor ions can optionally be selected for fragmentation in MS / MS applications.

[0106] In some preferred embodiments of the conjugate, the conjugate has a molecular weight in the range of ≥500 g / mol, preferably ≥700 g / mol, and / or an M + peak in the mass spectrum with m / z ≥500, preferably ≥700. The molecular weight of the conjugate is preferably high enough such that the M + peak generated in the mass spectrum is outside the range of the low molecular weight background, i.e., the molecular weight of the derivatizing agent (which is conjugated to the target analyte) is high enough such that the M + peak of the conjugate is outside the low molecular weight background range.

[0107] In some preferred embodiments of the conjugate, the conjugate comprises a neutral loss unit C-L1-Z, wherein C, L1 and Z are as defined above, which has a molecular weight of ≥ 300 g / mol and / or a peak at m / z ≥ 300 in the mass spectrum, preferably a molecular weight of ≥ 320 g / mol and / or a peak at m / z ≥ 320 in the mass spectrum, more preferably a molecular weight of ≥ 350 g / mol and / or a peak at m / z ≥ 350 in the mass spectrum, more preferably a molecular weight of ≥ 370 g / mol and / or a peak at m / z ≥ 370 in the mass spectrum, more preferably a molecular weight of ≥ 380 g / mol and / or a peak at m / z ≥ 380 in the mass spectrum.

[0108] The term "neutral loss unit" refers to a unit that is capable of losing an uncharged entity, i.e., it is capable of releasing a neutral entity. Generally, the neutral entity comprises a single atom or multiple atoms. The neutral loss unit can be neutral, positively charged or negatively charged. Under the conditions of MS, the neutral loss unit is capable of fragmenting, thereby releasing at least one neutral entity. After releasing the neutral entity, the remainder of the neutral loss unit retains its original charge. Accordingly, in the case where the neutral loss unit is uncharged, it remains neutral after losing the neutral entity. In the case where the neutral loss unit is positively charged, it remains positively charged after losing the neutral entity. In the case where the neutral loss unit is negatively charged, it remains negatively charged after losing the neutral entity. Generally, the release of the neutral entity occurs in a single fragmentation event. The term "fragmentation" refers to the dissociation of a single molecule into two or more independent molecules. As used herein, the term fragmentation refers to a specific fragmentation event, wherein the break point in the parent molecule where the fragmentation event occurs is well defined, and wherein the two or more sub-molecules resulting from the fragmentation event are well characterized. How to determine the break point of the parent molecule and the two or more resulting sub-molecules is well known to those skilled in the art. The resulting sub-molecules can be stable or can dissociate in subsequent fragmentation events. Fragmentation can occur by collision-induced dissociation (CID), electron-capture dissociation (ECD), electron-transfer dissociation (ETD), negative electron-transfer dissociation (NETD), electron separation dissociation (EDD), photodissociation (especially infrared multiphoton dissociation (IRMPD) and blackbody infrared radiative dissociation (BIRD)), surface-induced dissociation (SID), high-energy C-trap dissociation (HCD), charge-remote fragmentation.

[0109] As described above, effective fragmentation is ensured by a specific combination of Z and N units, which in some embodiments is a quaternary amine group adjacent to the benzyl position, enabling a smooth and selective neutral loss fragmentation process. During MS / MS, the conjugate of the derivatizing reagent and the analyte fragments by releasing large fragments and producing ions (the target analyte modified with a benzyl cation). This large mass shift is also highly advantageous as it ensures low background / interference for this novel class of reagents, as this specific and sensitive neutral loss pathway via large molecular weight loss is very rare.

[0110] Aspect 5 - Method for mass spectrometry of an analyte molecule

[0111] In a fifth aspect, the present invention relates to a method for mass spectrometry of analyte molecules, the method comprising the steps of:

[0112] (a) providing a target analyte;

[0113] (b) providing a derivatizing agent comprising a structure of formula (I) as defined in the section related to the first aspect; (c) reacting the analyte provided according to (a) with the derivatizing agent provided according to (b), thereby forming a preferably covalently bound conjugate of the analyte and the derivatizing agent, and (d) subjecting the conjugate formed in (c) to mass spectrometry.

[0114] Preferably, the mass spectrometry is laser desorption ionization mass spectrometry (LDI-MS), more preferably (MA)LDI-MS, even more preferably (MA)LDI-MS / MS, or (SA)LDI-MS, more preferably (SA)LDI-MS / MS.

[0115] All details, embodiments, and preferred embodiments of the first, second, third, and fourth aspects of the present invention described in the above sections, particularly the details, embodiments, and preferred embodiments described in the section related to the first aspect of the present invention, also apply to the fifth aspect of the present invention.

[0116] The present invention is further illustrated by the following examples and combinations of examples indicated by the respective dependencies and cross-references. In particular, it should be noted that in each case where the scope of an example is mentioned, for example in the context of terms such as "according to any one of Examples 1 to 4", each example within that scope is intended to be explicitly disclosed to the person skilled in the art, i.e., the wording of the term should be understood by the person skilled in the art as being synonymous with "according to any one of Examples 1, 2, 3, and 4".

[0117] 1. A derivatizing agent, preferably for derivatizing analytes intended for analysis via laser desorption ionization mass spectrometry (LDI-MS), the derivatizing agent comprising a structural element of formula (I),

[0118] C–L1–Z–(L2) p –X (I)

[0119] wherein

[0120] C is a chromophore having an absorption maximum in the range of 280 to 400 nm;

[0121] Z is a charged unit that includes at least one permanently charged moiety;

[0122] X is a reactive group;

[0123] L1 and L2 are each a linker unit; and

[0124] p is zero or 1.

[0125] 2. The derivatizing agent according to Example 1, wherein the absorption maximum of the chromophore C is the absorption maximum determined by UV / VIS spectroscopy.

[0126] 3. The derivatizing agent according to Example 1 or 2, wherein the absorption maximum of the chromophore C is in the range of 290 nm to 380 nm, preferably in the range of 300 nm to 360 nm, more preferably in the range of 305 nm to 330 nm.

[0127] 4. The derivatizing agent according to any one of Examples 1 to 3, wherein the chromophore C has the structure of formula (C),

[0128]

[0129] wherein R 1 、R 2 、R 3 、R 4 、R 5 are independently selected from the group consisting of: a hydrogen atom; a hydroxyl group; an NR x R y group, wherein R x and R y are independently a hydrogen atom or a C1 to C5 alkyl group; C1 to C5 alkyl, C5 to C10 (hetero)aryl, and –O-C1 to C3 alkoxy groups; and R 6 is absent (i.e., there is a direct covalent single bond between the aromatic ring and the C(=O) group) or is a –CR 7 =CR 8 - group, wherein R 7 is a hydrogen atom or a C1 to C3 alkyl group and R 8 is selected from the group consisting of a hydrogen atom, a C1 to C5 alkyl group, and an electron-withdrawing group; and the dashed line represents the bond to the linker, and the bond is preferably a single bond.

[0130] 5. The derivatizing agent according to any one of Embodiments 1 to 4, wherein the chromophore C has a structure of formula (C1), (C2), or (C3):

[0131]

[0132] wherein R 1 , R 2 , R 3 , R 4 are independently selected from the group consisting of: a hydrogen atom, a hydroxyl group, an NR x R y group, wherein R x and R y are independently a hydrogen atom or a C1-C5 alkyl group, C1-C5 alkyl, C5-C10 (hetero)aryl, and –O-C1-C3 alkoxy group.

[0133] 6. The derivatizing agent according to Embodiment 5, wherein the chromophore C has a structure of formula (C1), wherein R 1 and R 4 are both methoxy groups and R 3 is a hydroxyl group (C1, where these residues R 2 , R 3 and R 4 are the remainder of sinapic acid).

[0134] 7. The derivatizing agent according to Embodiment 5, wherein the chromophore C has a structure of formula (C2), wherein R 2 and R 4 are both hydroxyl groups (C2, where these residues R 2 , R 4 are the remainder of 2,5-dihydroxybenzoic acid).

[0135] 8. The derivatizing agent according to Embodiment 5, wherein the chromophore C has a structure of formula (C3), wherein R 3 is a hydroxyl group (C3, where the residue R 3 is the remainder of α-cyano-4-hydroxycinnamic acid).

[0136] 9. The derivatizing agent according to any one of Embodiments 1 to 8, wherein the linker L1 is selected from the group consisting of: (C1-C5 alkylene-O-) mgroups, where m is an integer in the range of 1 to 10, C1-C20 alkylene groups, C1-C20 alkylene-heteroaryl groups, and (C1-C5 alkylene)-O-(C1-C5 alkylene) groups, optionally linked or crossed with units selected from the group consisting of (hetero)aryl groups, N2, NO, NO2, S2, SO, SO2, CO, and CO2, and if present, the unit is preferably a heteroaryl group, more preferably from triazole, phenyltriazole, tetrazole, and phenyltetrazole.

[0137] 10. The derivatizing agent according to any one of embodiments 1 to 9, wherein the linker L1 has the structure (L1a) q -(L1b) r -(L1c) s , where q, r, s are each zero or 1, provided that at least one of q, r, s is 1; L1a is selected from (C1-C5 alkylene-O-) m groups, where m is an integer in the range of 1 to 10, C1-C10 alkylene groups; L1b is a unit selected from the group consisting of N2, NO, NO2, S2, SO, SO2, CO, CO2, triazole, phenyltriazole, tetrazole, and phenyltetrazole, and the unit is preferably triazole or tetrazole, more preferably the unit N is triazole, more preferably the unit N is 1,2,3-triazole ring, which is bonded to the linker L1a (if present) via a single bond at position 1 (N atom) of the triazole ring and bonded to the linker L1c (if present) via a single bond at position 4 (C atom) of the triazole ring; L1c (if present) is selected from the group consisting of (C1-C5 alkylene-O-) m groups, where m is an integer in the range of 1 to 10, C1-C10 alkylene, and (C1-C5 alkylene)-O-(C1-C5 alkylene) groups.

[0138] 11. The derivatizing agent according to any one of embodiments 1 to 10, wherein the linker L1 has the structure where the dotted lines respectively represent the bonds to (C) and (Z).

[0139] 12. The derivatizing agent according to any one of embodiments 1 to 11, wherein the charged unit Z is positively charged or negatively charged, preferably positively charged.

[0140] 13. The derivatizing agent according to any one of embodiments 1 to 12, wherein the charged unit Z is positively charged and is preferably a tetraalkylammonium group, more preferably -CH2N + (CH3)2CH2- group.

[0141] 14. A derivatizing agent according to any one of embodiments 1 to 12, wherein the charged unit Z is negatively charged, and the negatively charged unit Z is preferably selected from the group consisting of phosphates, sulfates, sulfonates, and carboxylates.

[0142] 15. A derivatizing agent according to any one of embodiments 1 to 12, wherein the linker L2 contains 1 to 10 C atoms and optionally one or more heteroatoms.

[0143] 16. The derivatizing agent according to embodiment 15, wherein the linker L2 is a C1-C5-alkylene-C5 to C10 aromatic ring, which is preferably substituted with a C(=O) unit as a substituent of the aromatic ring, and the linker L2 is preferably a C1-C3-alkylene-C6 aromatic ring, which is preferably substituted with a C(=O) unit as a substituent of the aromatic ring, wherein the C6 aromatic ring is more preferably a substituted or unsubstituted benzene ring, more preferably a benzene ring, which has a C(=O) group at the position of the benzene ring, which is para to the position where it is bonded to the C1-C3-alkylene, and more preferably has no other substituents.

[0144] 17. A derivatizing agent according to any one of embodiments 1 to 16, wherein the reactive group X is selected from the group consisting of: a carbonyl-reactive unit, a diene-reactive unit, a hydroxyl-reactive unit, an amino-reactive unit, an imine-reactive unit, a thiol-reactive unit, a diol-reactive unit, a phenol-reactive unit, an epoxide-reactive unit, a disulfide-reactive unit, and an azide-reactive unit.

[0145] 18. A kit comprising a derivatizing agent according to any one of embodiments 1 to 17.

[0146] 19. Use of a derivatizing agent according to any one of embodiments 1 to 17 for mass spectrometry of an analyte molecule, wherein the mass spectrometry is laser desorption ionization mass spectrometry (LDI-MS), preferably (MA)LDI-MS, more preferably (MA)LDI-MS / MS, or (SA)LDI-MS, more preferably (SA)LDI-MS / MS.

[0147] 20. A conjugate of a derivatizing agent according to any one of embodiments 1 to 17 and an analyte, wherein the conjugate has the structure of formula (II),

[0148] C–L1–Z–(L2) p –Xa–Ya-A (II)

[0149] wherein C, L1, L2, p, Z and N are as defined in any one of Examples 1 to 17; Xa is the remainder of the reactive group X as defined in any one of Examples 1 to 17; A is the analyte and Ya is the remainder of the reactive group Y bound to the analyte A, which has reacted with the reactive group X of the derivatizing agent, thereby forming a covalent bond between Xa and Ya.

[0150] 21. The conjugate according to Example 20, wherein the analyte is selected from the group consisting of nucleic acids (preferably selected from DNA, mRNA, miRNA and rRNA), amino acids, peptides, proteins (preferably cell surface receptors or cytosolic proteins), metabolites, hormones (preferably selected from testosterone, estrogen and estradiol), fatty acids, lipids, carbohydrates, steroids, ketosteroids, secosteroids (vitamin D), certain modified molecular features of another molecule (preferably selected from sugar moieties, phosphoryl residues on proteins, methyl residues on genomic DNA), substances internalized in vivo (preferably selected from therapeutic drugs, abused drugs, toxins) and metabolites of such substances.

[0151] 22. The conjugate according to Example 20 or 21, wherein the reactive group Y is selected from the group consisting of carbonyl groups, diene groups, hydroxyl groups, amine groups, imine groups, thiol groups, diol groups, phenol groups, epoxide groups, disulfide groups and azide groups.

[0152] 23. The conjugate according to any one of Examples 20 to 22, having a molecular weight in the range of ≥500 g / mol, preferably ≥700 g / mol and / or an M + peak in the mass spectrum with m / z ≥500, preferably ≥700.

[0153] 24. The conjugate according to any one of Examples 20 to 22, comprising a neutral loss unit C-L1-Z having a molecular weight of ≥300 g / mol and / or a peak in the mass spectrum with m / z ≥300, preferably ≥320 g / mol and / or a peak in the mass spectrum with m / z ≥320, more preferably ≥350 g / mol and / or a peak in the mass spectrum with m / z ≥350, more preferably ≥370 g / mol and / or a peak in the mass spectrum with m / z ≥370, more preferably ≥380 g / mol and / or a peak in the mass spectrum with m / z ≥380.

[0154] 25. A method for mass spectrometry of analyte molecules,

[0155] The method comprising the steps of:

[0156] (e) Provide a target analyte;

[0157] (f) Provide a derivatizing agent comprising a structure of formula (I) as defined in any one of Examples 1 to 17;

[0158] (g) React the analyte provided according to (a) with the derivatizing agent provided according to (b), thereby forming a conjugate preferably covalently bound between the analyte and the derivatizing agent, and

[0159] (h) Perform mass spectrometry on the conjugate formed in (c).

[0160] 26. A method for mass spectrometry, wherein the mass spectrometry is laser desorption ionization mass spectrometry (LDI-MS), preferably (MA)LDI-MS, more preferably (MA)LDI-MS / MS, or (SA)LDI-MS, more preferably (SA)LDI-MS / MS.

[0161] The present invention is further illustrated by the following reference examples, comparative examples and examples.

[0162] Example (Comparative) Example 1: Synthesis of [4-(hydrazinocarbonyl)phenyl]-N,N,N-trimethylammonium bromide (Alkyne 3)

[0163]

[0164] Dissolve bromide 1 (2.00 g, 8.73 mmol) and N,N-dimethylpropargylamine (4.8 ml, 64.1 mmol) in 30 ml of EtOH, and stir the reaction mixture at room temperature for 16 h. Collect the precipitate by filtration, wash with EtOH and Et2O and dry in vacuo. The desired product 2 was obtained as a white solid (2.73 g, quantitative yield).

[0165] 1 1H-NMR (400 MHz, methanol-d4): δ [ppm] = 3.19 (s, 3H), 3.68 (t, 1H), 3.93 (s, 3H), 4.31 (d, 2H), 4.74 (s, 2H), 7.75 (d, 2H), 8.16 (d, 2H).

[0166] 13 13C-NMR (101 MHz, methanol-d4): δ [ppm] = 49.47, 51.56, 53.43, 65.89, 71.10, 82.48, 129.87, 131.72, 132.37, 132.90, 166.08.

[0167] ESI-MS: 232.2 ([M + ​+ , Calculated value: 232.3)

[0168] Hydrazine monohydrate (6.8 ml, 64.1 mmol) was added to a solution of tertiary amide 2 (2.00 g, 6.41 mmol) in 30 ml of MeOH and the reaction mixture was stirred at room temperature. After 16 h, the reaction mixture was concentrated in vacuo and the crude product was purified by preparative RP-HPLC using an isocratic mobile phase (100% water). The desired product 3 was obtained as a yellow oil (1.69 g, 5.41 mmol, 84%).

[0169] 1 1H-NMR: (400 MHz, methanol-d4): δ [ppm] = 3.19 (s, 3H), 3.68 (t, 1H), 4.28 (d, 2H), 4.71 (s, 2H), 7.58 (d, 2H), 7.86 (d, 2H).

[0170] 13 13C-NMR (101 MHz, methanol-d4): δ [ppm] = 42.13, 49.41, 53.32, 60.70, 65.97, 71.13, 82.44, 127.72, 132.86, 133.96, 134.78, 168.21.

[0171] ESI-MS: 232.2 ([M+]+, calculated value: 232.3).

[0172] Example 1: Synthesis of derivatizing agent 5a

[0173]

[0174] Under an argon atmosphere, a solution of alkyne 3 (310 mg, 1.0 mmol) and azide 4 (101 mg, 0.33 mmol) in THF / water (2 ml, 1 / 1 (v / v)) was added to a solution of CuBr (16 mg, 0.10 mmol) and THPTA (44 mg, 0.10 mmol) in THF / water (2 ml, 1 / 1 (v / v)). The reaction mixture was stirred at room temperature for 24 h and then dried in vacuo. The crude product was purified by preparative RP-HPLC using a linear gradient from water / acetonitrile 100 / 0 -> 0 / 100 over 60 min. The desired product 5a was obtained as a beige solid (90 mg, 0.15 mmol, 44%) after lyophilization.

[0175] 11H-NMR: (400 MHz, DMSO-d6): δ [ppm] = 2.05 (m, 3H), 2.91 (m, 5H), 3.18 (m, 3H), 3.76 (m, 5H), 4.57 (m, 5H), 6.51 (d, 1H), 6.82 (s, 1H), 7.28 (d, 1H), 7.75 (d, 2H), 7.94 (d, 2H), 8.27 (m, 1H), 8.53 (m, 1H)

[0176] ESI-MS: 538.5 ([M+]+, calculated value: 538.6).

[0177] Example 2: Synthesis of the conjugate 6a by coupling the derivatizing agent 5a with the analyte (testosterone)

[0178]

[0179] Hydrazide 5a (55 mg, 90.0 μmol) and testosterone (77 mg, 270 μmol) were dissolved in 1 mL of methanol / formic acid (99 / 1, v / v). The reaction mixture was stirred at room temperature for 16 h and then dried in vacuo. The crude product was purified by preparative RP-HPLC using a linear gradient from water / acetonitrile 100 / 0 -> 0 / 100 over 60 min. After lyophilization, the desired hydrazide 6a was obtained as a beige solid (35.6 mg, 40.0 μmol, 44%).

[0180] 1 1H-NMR: (400 MHz, methanol-d4): δ [ppm] = 0.50 - 2.45 (m, 27H), 2.91 (m, 5H), 3.17 (m, 3H), 3.76 (s, 4H), 4.47 (m, 2H), 4.59 (m, 2H), 4.66 (m, 1H), 5.60 (s, 1H), 6.49 (d, 1H), 6.81 (s, 2H), 7.28 (d, 1H), 7.72 (d, 2H), 7.94 (d, 2H), 8.28 (s, 1H), 8.50 (m, 1H).

[0181] ESI-MS: 808.5 ([M+]+, calculated value: 808.5).

[0182] Example 3: Synthesis of the derivatizing agent 5b

[0183]

[0184] Under an argon atmosphere, a solution of alkyne 3 (310 mg, 1.0 mmol) and azide 4b (111 mg, 0.33 mmol) in THF / water (2 ml, 1 / 1 (v / v)) was added to a solution of CuBr (16 mg, 0.10 mmol) and THPTA (44 mg, 0.10 mmol) in THF / water (2 ml, 1 / 1 (v / v)). The reaction mixture was stirred at room temperature for 24 h and then dried in vacuo. The crude product was purified by preparative RP-HPLC within 60 min using a linear gradient from water / acetonitrile 100 / 0 -> 0 / 100. After lyophilization, the desired product 5b was obtained as a beige solid (138 mg, 0.21 mmol, 64%).

[0185] ESI-MS: 568.4 ([M+]+, calcd: 568.6).

[0186] Example 4: Coupling of derivatizing agent 5b with analyte (testosterone) - synthesis of conjugate 6b

[0187]

[0188] Hydrazide 5b (115 mg, 0.18 mmol) and testosterone (153 mg, 0.53 mmol) were dissolved in 1 ml of methanol / formic acid (99 / 1, v / v). The reaction mixture was stirred at room temperature for 16 h and then dried in vacuo. The crude product was purified by preparative RP-HPLC within 60 min using a linear gradient from water / acetonitrile 100 / 0 -> 0 / 100. After lyophilization, the desired hydrazide 6b was obtained as a beige solid (54.0 mg, 58.8 μmol, 32%).

[0189] 1 1H-NMR: (400 MHz, methanol-d4): δ [ppm] = 0.56 - 2.40 (m, 20H), 2.89 (s, 3H), 3.76 (s, 3H), 3.83 (m, 1H), 4.60 (m, 3H), 6.52 (m, 1H), 6.83 (s, 1H), 7.28 (d, 1H), 7.70 (m, 2H), 7.95 (m, 2H), 8.12 (s, 1H), 8.47 (m, 2H).

[0190] ESI-MS: 838.5 ([M+]+, calcd: 838.5).

[0191] Comparative Example 2: Coupling of derivatizing agent 3 with analyte (testosterone) - synthesis of conjugate 6c

[0192]

[0193] Alkyne 3 was reacted with testosterone (153 mg, 0.53 mmol) to afford conjugate 6c.

[0194] Example 5: Laser Desorption / Ionization Mass Spectrometry

[0195] 5a Sample Preparation and General Measurement Details

[0196] Stock solutions of all relevant analytes 6a, 6b, 6c, and testosterone were prepared separately at a concentration of 1.0 mg / ml (100% acetonitrile; ACN) and further diluted to achieve individual molar concentrated solutions of 12 μM (80 / 10 = ACN / H2O; abbreviated as 80% ACN). Analyte mixtures were prepared from the molar concentrated stock solutions to obtain a molar concentration of 3 μM (80% ACN) for each analyte. Further dilutions of the analyte mixture solution were prepared at 300 nM, 30 nM, and 3.0 nM (80% ACN).

[0197] For sinapic acid matrix experiments, the matrix solution was freshly prepared and consisted of 10 mg / ml sinapic acid in 50% ACN, 0.1% formic acid (FA).

[0198] As the MALDI target plate, a common 96-well steel plate (Waters Corp.) was used. Similarly, the SALDI-MS target plate was also based on a steel plate but had a functional amorphous a-C:H:Si:X (X = heteroatom-modified) plasma-activated chemical vapor deposition (PACVD) surface coating as the top layer.

[0199] All subsequent laser desorption / ionization mass spectrometry (LDI-MS) measurements were carried out in positive ion mode on a MALDI Synapt G2-Si (Waters Corp.) MALDI-Q-ToF mass spectrometer. For MALDI experiments, the laser repetition rate of the LDI-MS system was set to 1.0 kHz, and for matrix-free SALDI experiments, it was set to 200 Hz, using a Nd:YAG laser wavelength of 355 nm. During laser irradiation, a plate movement of 12 Hz was utilized, and each analyte spot was measured by a random path over the entire analyte spot area. The total acquisition time for each measurement was set to 30 s, with a mass spectrometry scan time of 0.5 s. The laser intensity could vary on a relative scale up to 500 – similar to a maximum output energy of 30 μJ – while the optimal laser energy for sinapic acid MALDI experiments was found to be 280 units, and the optimal laser energy for matrix-free SALDI experiments increased to 380 units. The individual voltage settings are listed in detail together with the corresponding measured values.

[0200] The corresponding data analysis was performed using the MassLynx 4.2 (SCN983, Waters Corp.) mass spectrometry instrument software. All the acquired mass spectrometry functions of each sample spot were accumulated during the entire 30 s analysis time.

[0201] Acquisition parameters for MALDI measurements:

[0202]

[0203]

[0204] Acquisition parameters for the corresponding SALDI measurements:

[0205]

[0206] 5b: Evaluation of derivatization reagents in matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS)

[0207] First, the applicability of the isolated derivatized testosterone conjugates 6a and 6b for possible matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) applications was tested. The performance of the two chromophore-containing conjugates 6a and 6b was compared with that of natural testosterone (T) and the derivatized testosterone conjugate 6c, which was used as the gold standard (in the field of liquid chromatography-based MS applications), but did not contain a chromophore suitable for LDI. An equimolar four-analyte mixture (containing all four analytes 6a, 6b, 6c, and testosterone (T)) was prepared at different concentrations (3 μM, 300 nM, 30 nM, and 3 nM), and then pre-mixed (1:1) with the sinapic acid matrix solution. Next, 1 μl of this analyte-matrix solution was applied to the MALDI target steel plate by dry-droplet preparation. The obtained analyte-matrix crystals were measured by MALDI-MS in the full-scan positive ion mode (m / z 200 to m / z 1000; for more details, see the experimental section). As expected, the analysis of the full-scan MALDI mass spectrum (see Figure 1 ) showed that the m / z signal intensity corresponding to the native analyte ( + , protonated testosterone cation) was very low. In contrast, the derivatized testosterone conjugates 6a and 6b were detected to have significantly higher intensities (about 96-fold signal enhancement, see Figure 2 ). Compared with the conjugate 6c of the prior art, it was clearly seen that the new reagent produced detectable ions with m / z that were not in the low molecular weight matrix background (m / z = 200 - 700 in this experiment), and thus were not susceptible to matrix-based interference. This solves a common problem of MALDI-MS (Anal Bioanal Chem 410, 4015–4038 (2018)).

[0208] Figure 2 Shows the comparison of the derivatized compounds [6a], + + with non-derivatized testosterone [TH] in the presence of sinapinic acid MALDI matrix + , [6b] + and [6c] + A dilution series of an equimolar (3 μM, 300 nM, 30 nM, and 3 nM) analyte mixture consisting of. The number of detected counts is shown on a logarithmic scale relative to the corresponding molar concentration.

[0209] However, compared to the prior art conjugate 6c with a concentration below 300 nM, the chromophore-bearing conjugates 6a and 6b in sinapinic acid did not result in signal enhancement. This observation indicates that in the presence of a suitable matrix (sinapinic acid in this case), the energy transfer from the laser beam to the target analyte is mediated via the matrix rather than via the derivatization reagent equipped with a chromophore. Considering the large excess of matrix molecules relative to the derivatized analyte, this is decisive. In summary, the chromophore-equipped derivatization reagents described herein provide a major "mass tag" advantage for MALDI-MS applications, which allows the detection of low molecular weight analytes in the absence of matrix-induced low molecular weight interferences.

[0210] 5c: Evaluation of Derivatization Reagents in Laser Desorption / Ionization Mass Spectrometry (LDI-MS) on Functionally Coated SALDI Plates

[0211] In accordance with the intent of the overall concept design, the laser desorption / ionization mass spectrometry (MALDI-MS) applications of the isolated derivatized testosterone conjugates 6a and 6b were evaluated. Again, the performance of the two chromophore-containing conjugates 6a and 6b was compared with that of natural testosterone (T) and the derivatized testosterone conjugate 6c without a suitable chromophore for LDI. An equimolar four-analyte mixture (containing all four analytes 6a, 6b, 6c, and testosterone (T)) at different concentrations (3000 nM, 300 nM, 30 nM, and 3 nM) was prepared and directly applied to a functionalized SALDI-MS target plate by dry-drop preparation. The spots obtained were measured by SALDI-MS in the full-scan positive ion mode (m / z 50 to m / z 1000; for more details, see the experimental section). As expected, the analysis of the full-scan LDI mass spectra (see the example at a concentration of 3 μM in Figure 3 ) showed that the m / z counts of the derivatized testosterone conjugates 6a and 6b increased compared to the natural analyte ([TH] + , protonated testosterone cation), corresponding to a signal enhancement of approximately 15-fold at a concentration of 3 μM.

[0212] Figure 4Shows in the presence of sinapic acid MALDI matrix with non-derivatized testosterone [TH] + Compared to + by the derivatized compounds [6a] + , [6b] + and [6c]

[0213] A dilution series of an equimolar (3 μM, 300 nM, 30 nM, and 3 nM) analyte mixture consisting of. The number of detected counts is shown on a logarithmic scale relative to the corresponding molar concentration. + This signal enhancement is particularly beneficial for high-sensitivity measurements at low concentrations. For example, natural testosterone cannot be detected at 3 nM, but can be detected at this low concentration after derivatization with reagents 5a and 5b (such as [6a] + and [6b] + ). Notably, compared to the prior art conjugate 6c lacking an LDI-compatible chromophore, the use of the LDI-compatible derivatization reagents 5a and 5b helps to increase the signal of the corresponding testosterone conjugates (such as [6a] + and [6b] + ). Compared to [6c] + , the absolute numbers of detected counts for [6a] + and [6b] + are 1.4-fold and 2.1-fold higher, respectively. More importantly, compared to [6c] + (S / N = 2), the signal-to-noise ratios of [6a] + (S / N = 304) and [6b]

[0214] Example 6 Generalization of the chromophore

[0215] Derivatization reagents 5a to 5c characterized by UV spectroscopy. The sinapic acid-derived reagents 5a and 5b showed absorption maxima at 307 nm and 315 nm, respectively, which are still comparable to those of conventionally used sinapic acid. Since the absorption maxima of these two compounds are relatively close (<40 nm) to the wavelength of the laser beam (355 nm), effective energy transfer can be expected. This clearly indicates that 5a and 5b are suitable for LDI applications because they are able to absorb the energy of the laser beam (usually 355 nm (J MassSpectrom. 2021; 56:4664)), which is considered a fundamental factor for MALDI matrices (Chem. Rev. 2003, 103, 2, 395–426). The local absorption maximum of reagent 5c is 240 nm, which is more than 100 nm lower than the wavelength of the conventionally used Nd:YAG laser. Based on our understanding and available data, this results in less spectral overlap with the laser beam and thus lower desorption / ionization efficiency compared to 5a and 5b.

[0216] Other small molecules (i.e., 2,5-dihydroxybenzoic acid and α-cyano-4-hydroxycinnamic acid) known to have similar local absorption maxima but different chemical structures (compared to sinapic acid) are also suitable for the (MA)LDI process (see Table 1). From these data, it is concluded that the proposed concept is not limited to structures 5a and 5b, but is applicable to any other derivatization reagent equipped with a chromophore C that is a suitable LDI chromophore.

[0217] Table 1 UV / VIS absorption maxima

[0218] Description of the Drawings

[0219] Figure 1 A full-scan MALDI mass spectrum (m / z 200 to m / z 1000) of an equimolar (3 μM) analyte mixture consisting of derivatized compounds 6a, 6b, 6c and natural testosterone in a sinapic acid MALDI matrix is shown.

[0220] Figure 2 Shows the dilution series of an equimolar (3 μM, 300 nM, 30 nM and 3 nM) analyte mixture consisting of derivatized compounds [6a] + +, [6b] + , [6c] + and [6c] + in the presence of a sinapic acid MALDI matrix compared to non-derivatized testosterone [TH]. The number of detected counts is shown on a logarithmic scale relative to the corresponding molar concentration.

[0221] Figure 3 Shows the comparison with non-derivatized testosterone [TH]+ In contrast, a full-scan SALDI mass spectrum (m / z 50 to m / z 1000) of an equimolar (3 μM) analyte mixture consisting of derivatized compounds [6a] + , [6b] + and [6c] + was acquired without the use of an additional MALDI matrix. *: [6a] + , [6b] + and [6c] + in-source fragmentation.

[0222] Figure 4 Shows a dilution series of an equimolar (3 μM, 300 nM, 30 nM, and 3 nM) analyte mixture consisting of derivatized compounds [6a] + , [6b] + , [6c] + and [6c] + compared to non-derivatized testosterone [TH]

[0223] Cited references

[0224] Higashi et al. (2016) J. of Pharmaceutical and Biomedical Analysis 130 pp. 181 - 190

[0225] WO 2011 / 091436 A1

[0226] WO 2011 / 091436 A1

[0227] Rahimoff et al. (2017) J. Am. Chem. Soc. 139(30), pp. 10359 - 10364

[0228] J. Sep. Sci. 2016, 39, 102–114; Biomed. Chromatogr. 2011; 25:1–10

[0229] Trends in Analytical Chemistry 143(2021)116399;

[0230] J Mass Spectrom. 2021; 56:e4731

[0231] J. Sep. Sci. 2016, 39, 102–114

[0232] Biomed.Chromatogr.2011;25:1–10

[0233] Anal.Chem.2020,92,6224-6228

[0234] Chem Asian J.2021,16,868–878

[0235] Crit Rev Anal Chem.Dec 30, 2021;1-17

[0236] Mass Spec Rev.2019;38:3–21

[0237] Scientific Reports|5:17853,ChemBioChem 2021,22,1430–143

[0238] International Journal of Mass Spectrometry 353(2013)54–59

[0239] Anal.Chem.2014,86,21,10724–10731

[0240] T.Higashi J Steroid Biochem Mol Biol.Sep 2016;l62:57-69

[0241] H.Ban et al.J.Am.Chem.Soc.,2010,132(5),pp.1523-1525

[0242] Anal.Bioanal.Chem.2011,399,2597,DOI 10.1007 / s00216-010-4063-3

[0243] Anal Bioanal Chem.2018,410,4015–4038

[0244] J Am Soc Mass Spectrom 2007,18,9,1582–1590

Claims

1. A derivatizing agent, preferably for an analyte intended for analysis by laser desorption ionization mass spectrometry (LDI-MS), said derivatizing agent comprising a structural element of formula (I), C–L1–Z–(L2) p –X(I) wherein C is a chromophore having an absorption maximum in the range from 280 to 400 nm; Z is a charged unit which comprises at least one permanently charged moiety; X is a reactive group; L1 and L2 are each a linker unit; and p is zero or 1.

2. The derivatizing agent according to claim 1, wherein the absorption maximum of the chromophore C is the absorption maximum determined by UV / VIS spectroscopy.

3. The derivatizing agent according to claim 1 or 2, wherein the chromophore C has a structure of formula (C), wherein R 1 , R 2 , R 3 , R 4 , R 5 are independently selected from the group consisting of: a hydrogen atom; a hydroxyl group; NR x R y group, wherein R x and R y are independently a hydrogen atom or a C1-C5 alkyl group; a C1-C5 alkyl, a C5-C10 (hetero)aryl, and an -O-C1-C3 alkoxy group; and R 6 is absent or is -CR 7 =CR 8 - group, wherein R 7 is a hydrogen atom or a C1-C3 alkyl group and R 8 is selected from the group consisting of a hydrogen atom, a C1-C5 alkyl group, and an electron-withdrawing group; and the dashed line represents a bond to the linker, and the bond is preferably a single bond.

4. The derivatizing agent according to any one of claims 1 to 3, wherein the linker L1 is selected from the group consisting of: (C1-C5 alkylene-O-) m a group, where m is an integer in the range of 1 to 10, a C1 to C20 alkylene group, a C1 to C20 alkylene-heteroaryl group, and a (C1-C5 alkylene)-O-(C1-C5 alkylene) group, optionally linked or crossed with a unit selected from the group consisting of (hetero)aryl groups, N2, NO, NO2, S2, SO, SO2, CO, and CO2.

5. The derivatizing agent according to any one of claims 1 to 4, wherein the charged unit Z is positively charged or negatively charged.

6. The derivatizing agent according to any one of claims 1 to 5, wherein the linker L2 comprises from 1 to 10 carbon atoms and optionally one or more heteroatoms.

7. The derivatizing agent according to any one of claims 1 to 6, wherein the reactive group X is selected from the group consisting of a carbonyl-reactive unit, a diene-reactive unit, a hydroxy-reactive unit, an amino-reactive unit, an imine-reactive unit, a thiol-reactive unit, a diol-reactive unit, a phenol-reactive unit, an epoxide-reactive unit, a disulfide-reactive unit, and an azide-reactive unit.

8. A kit which comprises the derivatizing agent according to any one of claims 1 to 7.

9. Use of the derivatizing agent according to any one of claims 1 to 7 for mass spectrometry of analyte molecules, wherein the mass spectrometry is laser desorption ionization mass spectrometry (LDI-MS), preferably (MA)LDI-MS, more preferably (MA)LDI-MS / MS or (SA)LDI-MS.

10. A conjugate of a derivatizing agent according to any one of claims 1 to 7 and an analyte, wherein the conjugate has a structure of formula (II), C–L1–Z–(L2) p –Xa–Ya-A(II) wherein C, L1, L2, p, Z and N are as defined in any one of claims 1 to 7; Xa is the remainder of the reactive group X as defined in any one of claims 1 to 7; A is the analyte and Ya is the remainder of the reactive group Y bound to the analyte A, which has reacted with the reactive group X of the derivatizing agent, thereby forming a covalent bond between Xa and Ya.

11. The conjugate according to claim 10, wherein the analyte is selected from the group consisting of nucleic acids, amino acids, peptides, proteins, metabolites, hormones, fatty acids, lipids, carbohydrates, steroids, ketosteroids, seco-steroids, molecules characterized by some modification of other molecules, substances internalized by an organism, and metabolites of such substances.

12. The conjugate according to claim 10 or 11, wherein the reactive group Y is selected from the group consisting of: a carbonyl group, a diene group, a hydroxyl group, an amine group, an imine group, a thiol group, a diol group, a phenol group, an epoxide group, a disulfide group, and an azide group.

13. The conjugate according to any one of claims 10 to 12, having a molecular weight in the range of ≥500 g / mol and / or an M peak with m / z ≥500 in the mass spectrum and / or comprising a neutral loss unit C-L1-Z having a molecular weight of ≥300 g / mol and / or a peak with m / z ≥300 in the mass spectrum. + Peak and / or comprising a neutral loss unit C-L1-Z having a molecular weight of ≥300 g / mol and / or a peak with m / z ≥300 in the mass spectrum.

14. A method for mass spectrometry of an analyte molecule, the method comprising the steps of: (a) providing a target analyte; (b) providing a derivatizing agent comprising a structure of formula (I) as defined in any one of claims 1 to 7; (c) reacting the analyte provided in (a) with the derivatizing agent provided in (b), thereby forming a preferably covalently bound conjugate of the analyte and the derivatizing agent, and (d) subjecting the conjugate formed in (c) to mass spectrometry, wherein the mass spectrometry is preferably laser desorption ionization mass spectrometry (LDI-MS), more preferably (MA)LDI-MS, more preferably (MA)LDI-MS / MS, or (SA)LDI-MS, more preferably (SA)LDI-MS / MS.

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