Patent Description:
The present disclosure belongs to the field of protein labeling reagents, and particularly relates to an isobaric stable isotope-containing phosphorylated protein labeling reagent, and a preparation method and application thereof.

Large-scale identification and quantitative analysis of proteins is a key to proteomics research. The new quantitative protein analysis technology plays a crucial role in the discovery of basic life mechanisms and disease markers, and the stable isotope chemical labeling technology based on biological mass spectrometry (bio-MS) plays an increasingly important role in the relative and absolute quantification of proteins[<NUM>]. The rapid development of bio-MS instruments plays an essential role in the application and research in protein identification, post-translational modification (PTM), and quantitative protein analysis. The development of bio-MS technology accelerates the progress of proteomics research, but the complexity of samples and the processing of massive data are inevitable challenges of bio-MS technology. It is believed that bio-MS technology will become more significant in proteomics research with the continuous improvement of bio-MS technology.

A variety of stable isotope-labeled protein MS (MS) quantification methods and reagents have been successfully developed; for example, iTRAQ[<NUM>], ICAT[<NUM>], and TMT[<NUM>] have been widely used as commercial reagents in life science and clinical research. Stable isotope-labeled molecular probes based on high-resolution MS have become a core for protein quantification in proteomics research. Various protein quantification methods have been successfully developed based on the principles of chemical reactions and instrumental enzyme catalytic labeling. For example, stable isotope tags based on primary MS, such as multi-labeled reductive methylation, <NUM>O labeling, SILAC (Stable Isotope Labeling by Amino Acids in Cell Culture), <NUM>Eu-labeled quantitative probe, and trypsin-catalyzed N-terminal arginine labeling. Recently, scientists have developed two brand-new techniques for quantifying isobaric labeled proteins: EASI-tag based on peptide reporter ions and TMTpro based on isopropyl-modified proline structures[<NUM>-<NUM>]. Stable isotope N-phosphoryl amino acids labeling strategy has been developed for quantitative profiling of amine-containing metabolites in urine based on organic phosphorus chemistry.

Although some innovations in reporter ion structures have been reported, no improvement has been made in the sensitivity of MS detection. In addition, the traditional labeling strategy is based on classical protein chemistry, selectivity of labeling chemistry, MS sensitivity and chromatographic separation of labeling reagents, isotope effects, high prices of reagents, and the like, and the quantitative flux is limited, which seriously restricts the study of large-scale quantitative analysis of proteomics. The development of new labeling strategies and labeling reagents is of great significance. Based on the organophosphorus labeling chemistry, the present disclosure synthesizes a series of isobaric stable isotope-labeled organophosphorus reagents with novel structures and uses the reagents to successfully achieve the quantitative analysis of standard proteins and proteins in biological samples.

In view of the shortcomings of the prior art, the present disclosure is intended to provide an isobaric stable isotope-containing phosphorylated protein labeling reagent, and a preparation method and application thereof. In order to achieve the above objective, the present disclosure adopts the following solutions:.

The present disclosure provides an isobaric stable isotope-containing phosphorylated protein labeling reagent, characterized in that its structural formula is as follows:
<CHM>
where a is <NUM> or <NUM>; b is <NUM> or <NUM>; c is <NUM> or <NUM>; d is <NUM> or <NUM>; n is <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> and preferably <NUM> or <NUM>; n<NUM> is <NUM>, <NUM>, or <NUM> and preferably <NUM> or <NUM>; R<NUM> is a side chain substituent of an L or D-configuration amino acid with or without a stable isotope, and preferably R<NUM> is methyl; and R<NUM> is a side chain substituent of an amino acid with or without a stable isotope, and preferably R<NUM> is hydrogen.

The present disclosure provides a preparation method of an isobaric stable isotope-containing phosphorylated protein labeling reagent, characterized in that it includes the following steps:.

Preferably, , the alkali solution in the steps (<NUM>) and (<NUM>) was one selected from the group consisting of a sodium hydroxide solution, a potassium hydroxide solution, a sodium carbonate solution, and a sodium bicarbonate solution and was preferably a sodium carbonate solution; the cooling and the low temperature were ice bath conditions; and the low-temperature stirring reaction time was for <NUM> to <NUM> and the room temperature stirring reaction was for <NUM> to <NUM>.

Preferably, the organic solvent in the steps (<NUM>) and (<NUM>) was one selected from the group consisting of ethylene glycol dimethyl ether and carbon dichloride; and the condensation agent was one selected from the group consisting of DCC and EDCI.

Preferably, the low temperature in the step (<NUM>) was an ice bath condition; and the low-temperature stirring reaction time was for <NUM> to <NUM> and the reaction under stirring at the room temperature was conducted for <NUM> to <NUM>.

Preferably, in the step (<NUM>), the isobaric stable isotope-containing alcohol solvent was one selected from the group consisting of methanol, ethanol, propanol, butanol, and amyl alcohol and was preferably methanol or ethanol.

The present disclosure provides an application of the isobaric stable isotope-containing phosphorylated protein labeling reagent in the quantitative analysis of a polypeptide.

The present disclosure provides an application of the isobaric stable isotope-containing phosphorylated protein labeling reagent in the quantitative analysis of a standard protein or a protein in a cell.

The present disclosure provides an application of the isobaric stable isotope-containing phosphorylated protein labeling reagent in the quantitative analysis of a protein in a urine sample.

The present disclosure provides an application of the isobaric stable isotope-containing phosphorylated protein labeling reagent in the quantitative analysis of a protein in a blood sample.

The specific principle of the present disclosure is as follows:
The isobaric stable isotope-containing phosphorylated protein labeling reagent provided by the present disclosure can be used as an isobaric stable isotope-containing phosphorylated labeled molecular probe (iSIPL) to label the N-terminal and lysine side-chain amino selectively and efficiently of a peptide and then achieve the relative quantitative analysis of the peptide and protein by MS analysis, especially the report ions produced by secondary MS cleavage. At present, most of the quantitative information of bio-MS comes from a primary MS spectrum, that is, stable isotope tags are first introduced into the peptides produced by protein enzymolysis, and then the relative quantification of a protein is conducted by comparing the intensities of MS peaks labeled with stable isotopes in the primary MS spectrum. The method provided by the present disclosure is based on the selection of peptide parent ions. Compared with the primary MS, the secondary MS dramatically reduces a noise level of the secondary MS spectrum, that is, improves a signal-to-noise ratio of the secondary MS spectrum, thereby improving the accuracy and quantitative flux of peptide and protein quantification.

The principle is explained herein by taking the quantification method of an isobaric stable isotope six-labeled N-phosphorylated protein as an example:
<CHM>.

As shown in the above figure, after isotope labeling of atoms numbered <NUM> to <NUM> in the isobaric stable isotope-containing phosphorylated protein labeling reagent, stable isotopes in the labeling reagent are mainly distributed in a phosphate group of a reporter ion and an equilibrium linker arm. In order to make the reporter ion appear at a low molecular weight end and have prominent stability and the highest MS analysis sensitivity in the secondary MS cleavage, an ethoxyl structure is adopted for the phosphate group; considering the availability of the <NUM>C and <NUM>N labeling synthesis module, glycine at a relatively low price is adopted for the equilibrium linker arm in the labeling reagent; and the interference of peptide cleavage fragment ions to the reporter ion is investigated after labeling with different organophosphorus reagents. A labeling reagent that does not interfere with the reporter ion in the corresponding m/z range is selected.

The present disclosure has the following advantages:.

The present disclosure is further described in detail below in conjunction with embodiments. The specific mass, reaction time and temperature, process parameters, and the like of the embodiments are each only an Example in an appropriate range. An embodiment in which no specific technology or condition is specified shall be conducted in accordance with the technology or condition described in the literature in the art or in accordance with product instructions. The used reagents or instruments not specified with a manufacturer are conventional products that can be purchased from the market.

<FIG> shows a structural formula of the isobaric stable isotope-containing phosphorylated protein labeling reagent prepared by the present disclosure.

The general method for quantitative analysis of an iSIPL-labeled polypeptide and protein includes: an isobaric stable isotope-containing phosphorylated protein labeling reagent is prepared into a reaction solution, then a sample to be tested (which can be any one selected from the group consisting of a polypeptide, a standard protein sample, a protein sample in a cell, and a protein in an urine and blood sample) is added to allow a derivatization reaction, and a resulting product is concentrated, desalted, and then directly analyzed by LC-MS.

The conditions of quantitative analysis by MS are as follows:
The MS includes electrospray ionization-time-of-flight MS (ESI-TOF MS), electrospray ionization-orbitaltrap MS (ESI-Orbitrap MS), electrospray ionization-ion trap MS (ESI-IT MS), matrix-assisted laser-desorption ionization-time-of-flight MS (MALDI-TOF/TOF MS), or the like. ESI-TOF MS and ESI-Orbitrap MS are preferred.

The ion ionization mode is a positive ion mode.

The cleavage mode is collision-induced dissociation (CID), high-energy collision dissociation (HCD), or the like. The HCD mode is preferred.

The high-performance liquid chromatograph includes Easy-NanoLC1000 nanoflow liquid chromatograph and DIONEX Ultimate <NUM> high-performance liquid chromatograph.

Example <NUM> Preparation of a diethyl phosphite alanine-glycine dipeptide activated ester, specifically including the following steps:.

DEPH-L-Ala-Gly (C<NUM>H<NUM>N<NUM>O<NUM>P): <NUM>H NMR (<NUM>, CDCl<NUM>): δ= <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>) ppm. <NUM>C NMR (<NUM>, CDCl<NUM>): δ= <NUM>, <NUM>, <NUM> (dd, J<NUM>= <NUM>, J<NUM>= <NUM>), <NUM>, <NUM>, <NUM>, <NUM> (dd, J<NUM>= J<NUM>= <NUM>) ppm. <NUM>P NMR (<NUM>, CDCl<NUM>): δ= <NUM> ppm. ESI-MS: [M-H]-, m/z <NUM> (theoretical value: <NUM>, relative error: <NUM> ppm).

The DEPH-L-Ala-Gly (<NUM> mmol, <NUM>) obtained above, N-hydroxysuccinimide (<NUM> mmol, <NUM>), and DCC (<NUM> mmol, <NUM>) were dissolved in <NUM> of dichloromethane, and the reaction stirred at room temperature overnight; after the reaction was completed, the mixture was filtered, the filtrate was concentrated; and the solid product was recrystallized with ethyl acetate and n-hexane in a ratio of <NUM>:<NUM> to obtain the white solid compound (namely, the target product), which was denoted as DEPH-Z-Ala-Gly-NHS, which has a structure shown as follows:
<CHM>.

DEPH-L-Ala-Gly-NHS (C<NUM>H<NUM>N<NUM>O<NUM>P): <NUM>H NMR (<NUM>, CDCl<NUM>): δ= <NUM> (t, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>) ppm. <NUM>C NMR (<NUM>, CDCl<NUM>): δ= <NUM> (d, J= <NUM>), <NUM>, <NUM>, <NUM> (dd, J<NUM>= J<NUM>= <NUM>), <NUM>, <NUM>, <NUM>, <NUM> (d, J= <NUM>), <NUM> (dd, J<NUM>= J<NUM>= <NUM>) ppm. <NUM>P NMR (<NUM>, CDCl<NUM>): δ= <NUM> ppm.

Example <NUM> Preparation of two-labeled diethyl phosphite alanine-glycine dipeptide activated ester with stable isotopes of nitrogen-<NUM>, specifically including the following steps:.

L-N<NUM>-Ala (<NUM> mmol) was dissolved in a NaOH (<NUM>, <NUM>) aqueous solution and cooled to <NUM>. A solution of (Boc)<NUM>O (<NUM>, <NUM> mmol) in <NUM>,<NUM>-dioxane (<NUM>) was added dropwise to the NaOH aqueous solution, NaHCO<NUM> (<NUM>, <NUM> mmol) was added, and a reaction mixture was stirred overnight at room temperature; a reaction solution was evaporated under reduced pressure, then <NUM> of water was added, and the excess (Boc)<NUM>O was removed with EtOAc (<NUM> × <NUM>), cooled in an ice bath and then acidified with a KHSO<NUM> aqueous solution (<NUM>) to a pH of <NUM> to <NUM>. The aqueous layer was extracted with EtOAc (<NUM> × <NUM>). Finally, the combined extraction solution was washed with a saturated NaCl solution, dried with anhydrous MgSO4, and the solvent was concentrated under reduced pressure to obtain a white solid product Boc-N<NUM>-Ala (<NUM>, <NUM>%), which had a structure shown as follows:
<CHM>.

Boc-N<NUM>-Ala (C<NUM>H<NUM><NUM>NO<NUM>): <NUM>H NMR (<NUM>, MeOD) δ= <NUM> (q, J= <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (dd, J= <NUM>, <NUM>, <NUM>) ppm. <NUM>C NMR (<NUM>, MeOD) δ= <NUM>, <NUM> (d, J= <NUM>), <NUM>, <NUM> (d, J= <NUM>), <NUM>, <NUM> ppm. ESI-MS: m/z= <NUM> ([M-H]-, theoretical mass: <NUM>, relative error: <NUM> ppm).

At <NUM>, DCC (<NUM>, <NUM> mmol) was added to a EtOAc solution of Boc-N<NUM>-Ala (<NUM>, <NUM> mmol), N-hydroxysuccinimide (<NUM>, <NUM> mmol). and a resulting mixture was stirred for <NUM> at a low temperature and further stirred for <NUM> at room temperature. The impurity of DCU generated was removed by filtration, and the solvent was evaporated under reduced pressure; and pentane (<NUM>) was added to the residue, stirred for <NUM> under N<NUM> and then filtered to obtain BOC-N<NUM>-Ala-OSU, which would be directly used in the next reaction.

The BOC-N<NUM>-Ala-OSU (<NUM> mmol) synthesized was taken and dissolved in <NUM> of acetone and <NUM> of ethanol, slowly added dropwise to a NaHCO<NUM> (<NUM> mmol, <NUM>) aqueous solution of glycine (<NUM> mmol, <NUM>) at <NUM>; subjected to a reaction at room temperature for <NUM> after the dropwise addition was completed. After the reaction was stopped; a part of the solvent was concentrated under reduced pressure and removed , <NUM> of H<NUM>O was added to the residue, and shaken for thorough mixing. The aqueous layer was extracted with ethyl acetate (<NUM> × <NUM>), the residual aqueous solution was cooled to <NUM>, and pH was adjusted to about <NUM> with diluted hydrochloric acid (<NUM>). The acidic aqueous solution was extracted with ethyl acetate (<NUM> × <NUM>), and the organic layers were combined, washed with a saturated NaCl aqueous solution, dried with anhydrous MgSO<NUM>, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was recrystallized with petroleum ether and ethyl acetate in a ratio of <NUM>:<NUM> to obtain a white solid product BOC-N<NUM>-Ala-Gly (<NUM>, <NUM>%), which had a structure shown as follows:
<CHM>.

Boc-N<NUM>-Ala-Gly (C<NUM>H<NUM>N<NUM>NO<NUM>): <NUM>H NMR (<NUM>, MeOD) δ= <NUM> - <NUM> (m, <NUM>), <NUM> (q, J= <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (dd, J= <NUM>, <NUM>, <NUM>) ppm. <NUM>C NMR (<NUM>, MeOD) δ= <NUM>, <NUM>, <NUM>, <NUM>, <NUM> (d, J= <NUM>), <NUM>, <NUM>, <NUM> ppm. ESI-MS: m/z= <NUM> ([M-H]-, theoretical mass: <NUM>, relative error: <NUM> ppm).

Similarly, BOC-Ala (<NUM> mmol, <NUM>) and N<NUM>-Gly (<NUM> mmol, <NUM>) were subjected to a reaction to obtain a white solid product BOC-Ala-N<NUM>-Gly (<NUM>, <NUM>%), which had a structure shown as follows:
<CHM>.

Boc-Ala-N<NUM>-Gly (C<NUM>H<NUM>N<NUM>NO<NUM>): <NUM>H NMR (<NUM>, MeOD) δ= <NUM> (m, <NUM>), <NUM> (q, J= <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J= <NUM>, <NUM>) ppm. <NUM>C NMR (<NUM>, MeOD) δ= <NUM>, <NUM>, <NUM>, <NUM>, <NUM> (d, J= <NUM>), <NUM> (d, J= <NUM>), <NUM>, <NUM> ppm. ESI-MS: m/z= <NUM> ([M-H]-, theoretical mass: <NUM>, relative error: <NUM> ppm).

BOC-N<NUM>-Ala-Gly (<NUM> mmol, <NUM>) was dissolved in <NUM> of THF, <NUM> of concentrated hydrochloric acid was added, subjected to a reaction for <NUM> at room temperature under the protection of N<NUM>, the solvent was evaporated, the residue was dissolved in <NUM> of H<NUM>O; the aqueous layer was extracted with CH<NUM>Cl<NUM> (<NUM> × <NUM>), evaporated under reduced pressure to obtain a light-yellow oily substance N<NUM>-Ala-Gly, which would be directly used for the next reaction without further purification. Similarly, BOC-Ala-N<NUM>-Gly (<NUM> mmol, <NUM>) was subjected to a reaction to obtain a light-yellow oily liquid Ala-N<NUM>-Gly, which had a structure shown as follows:
<CHM>.

N<NUM>-Ala-Gly (C<NUM>H<NUM>N<NUM>NO<NUM>): <NUM>H NMR (<NUM>, D<NUM>O) δ= <NUM> (q, J= <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (dd, J= <NUM>, <NUM>, <NUM>) ppm. <NUM>C NMR (<NUM>, D<NUM>O) δ= <NUM>, <NUM>, <NUM> (d, J= <NUM>), <NUM>, <NUM> ppm. ESI-MS: m/z= <NUM> ([M-H]-, theoretical mass: <NUM>, relative error: <NUM> ppm).

Similarly, Ala-N<NUM>-Gly was prepared, which had a structure shown as follows:
<CHM>.

Ala-N<NUM>-Gly (C<NUM>H<NUM>N<NUM>NO<NUM>): <NUM>H NMR (<NUM>, D<NUM>O) δ= <NUM> (q, J= <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J= <NUM>, <NUM>) ppm. <NUM>C NMR (<NUM>, D<NUM>O) δ= <NUM>, <NUM> (d, J= <NUM>), <NUM> (d, J= <NUM>), <NUM> (d, J= <NUM>), <NUM> ppm. ESI-MS: m/z= <NUM> ([M-H]-, theoretical mass: <NUM>, relative error: <NUM> ppm).

N<NUM>-Ala-Gly (<NUM> mmol, <NUM>) was dissolved in <NUM> of water, then <NUM> of triethylamine and <NUM> of ethanol were added, thoroughly stirred and then cooled to <NUM>; diethyl phosphite (<NUM> mmol, <NUM>) was taken and dissolved in <NUM> of carbon tetrachloride, added dropwise to the dipeptide solution, subjected to a reaction for <NUM> at a low temperature; and after the reaction was stopped, a part of the solvent was evaporated under reduced pressure and removed, and the crude product was subjected to high-performance liquid chromatography to obtain a transparent oily substance DEPH-N<NUM>-Ala-Gly (<NUM>, <NUM>%). Similarly, Ala-N<NUM>-Gly (<NUM> mmol, <NUM>) and diethyl phosphite (<NUM> mmol, <NUM>) were subjected to a reaction to obtain a transparent oily substance DEPH-Ala-N<NUM>-Gly (<NUM>, <NUM>%), which had a structure shown as follows:
<CHM>.

DEPH-N<NUM>-Ala-Gly (C<NUM>H<NUM>N<NUM>NO<NUM>P): <NUM>H NMR (<NUM>, CDCl<NUM>) δ= <NUM> (s, <NUM>), <NUM> (dd, J= <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (dd, J= <NUM>, <NUM>, <NUM>), <NUM> (dd, J= <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>) ppm. <NUM>C NMR (<NUM>, CDCl<NUM>) δ= <NUM>, <NUM>, <NUM> (dd, J= <NUM>, <NUM>), <NUM> (d, J= <NUM>), <NUM>, <NUM> (d, J= <NUM>), <NUM> (dd, J= <NUM>, <NUM>). <NUM>P NMR (<NUM>, CDCl<NUM>) δ= <NUM> ppm. ESI-MS: m/z= <NUM> ([M + H]+, theoretical mass: <NUM>, relative error: <NUM> ppm), <NUM> ([M + Na]+, theoretical mass: <NUM>, relative error: <NUM> ppm).

Similarly, DEPH-Ala-N<NUM>-Gly was prepared, which had a structure shown as follows:
<CHM>.

DEPH-Ala-N<NUM>-Gly (C<NUM>H<NUM>N<NUM>NO<NUM>P): <NUM>H NMR (<NUM>, CDCl<NUM>) δ= <NUM> (dq, J= <NUM>, <NUM>, <NUM>), <NUM> (dd, J= <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (dd, J= <NUM>, <NUM>, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM> (dt, J= <NUM>, <NUM>, <NUM>) ppm. <NUM>C NMR (<NUM>, CDCl<NUM>) δ= <NUM> (dd, J= <NUM>, <NUM>), <NUM>, <NUM> (dd, J= <NUM>, <NUM>), <NUM> (d, J= <NUM>), <NUM> (d, J= <NUM>), <NUM> (d, J= <NUM>), <NUM> (dd, J= <NUM>, <NUM>) ppm. <NUM>P NMR (<NUM>, CDCl<NUM>) δ= <NUM> ppm. ESI-MS: m/z= <NUM> ([M + H]+, theoretical mass: <NUM>, relative error: <NUM> ppm), <NUM> ([M + Na]+, theoretical mass: <NUM>, relative error: <NUM> ppm).

DEPH-N<NUM>-Ala-Gly (<NUM> mmol, <NUM>) and N-hydroxysuccinimide (<NUM> mmol, <NUM>) were dissolved in <NUM> of dichloromethane, DCC (<NUM> mmol, <NUM>) was added at <NUM>, the reaction solution was subjected to a reaction for half an hour at a low temperature, transferred to a room temperature and the reaction stirred overnight; and after the reaction was completed, the mixture was filtered, the filtrate was concentrated, and the product was recrystallized with dichloromethane and n-hexane in a ratio of <NUM>:<NUM> to obtain the white solid compound DEPH-N<NUM>-Ala-Gly-NHS (<NUM>, <NUM>%). Similarly, DEPH-Ala-N<NUM>-Gly (<NUM> mmol, <NUM>) and N-hydroxysuccinimide (<NUM> mmol, <NUM>) were subjected to a reaction to obtain DEPH-Ala-N<NUM>-Gly-NHS (<NUM>, <NUM>%), which had a structure shown as follows:
<CHM>.

DEPH-N<NUM>-Ala-Gly-NHS (C<NUM>H<NUM>N<NUM><NUM>NO<NUM>P): <NUM>H NMR (<NUM>, CDCl<NUM>) δ= <NUM> (t, J= <NUM>, <NUM>), <NUM> (dd, J= <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (dt, J= <NUM>, <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (dd, J= <NUM>, <NUM>, <NUM>), <NUM> (dt, J= <NUM>, <NUM>, <NUM>) ppm. <NUM>C NMR (<NUM>, CDCl<NUM>) δ= <NUM> (d, J= <NUM>), <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> (dd, J= <NUM>, <NUM>), <NUM> (d, J= <NUM>), <NUM>, <NUM>, <NUM> (d, J= <NUM>), <NUM> (dd, J= <NUM>, <NUM>) ppm. <NUM>P NMR (<NUM>, CDCl<NUM>) δ= <NUM> ppm. ESI-MS: m/z= <NUM> ([M + H]+, theoretical mass: <NUM>, relative error: <NUM> ppm), <NUM> ([M + Na]+, theoretical mass: <NUM>, relative error: <NUM> ppm).

Similarly, DEPH-Ala-N<NUM>-Gly-NHS was prepared, which had a structure shown as follows:
<CHM>.

DEPH-Ala-N<NUM>-Gly-NHS (C<NUM>H<NUM>N<NUM><NUM>NO<NUM>P): <NUM>H NMR (<NUM>, CDCl<NUM>) δ= <NUM> (dt, J= <NUM>, <NUM>, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (dt, J= <NUM>, <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM> (dt, J= <NUM>, <NUM>, <NUM>) ppm. <NUM>C NMR (<NUM>, CDCl<NUM>) δ= <NUM> (dd, J= <NUM>, <NUM>), <NUM>, <NUM>, <NUM> (dd, J= <NUM>, <NUM>), <NUM> (d, J= <NUM>), <NUM> (d, J= <NUM>), <NUM>, <NUM> (d, J= <NUM>), <NUM> (dd, J= <NUM>, <NUM>) ppm. <NUM>P NMR (<NUM>, CDCl<NUM>) δ= <NUM> ppm. ESI-MS: m/z= <NUM> ([M + H]+, theoretical mass: <NUM>, relative error: <NUM> ppm), <NUM> ([M + Na]+, theoretical mass: <NUM>, relative error: <NUM> ppm).

<NUM>N-L-Ala (<NUM> mmol, <NUM>) was weighted and dissolved in <NUM> of a Na<NUM>CO<NUM> (<NUM> mmol, <NUM>) aqueous solution, cooled to <NUM>, and then <NUM> of <NUM>,<NUM>-dioxane was slowly added dropwise; <NUM>-fluorenylmethyl chloroformate (<NUM> mmol, <NUM>) was dissolved in <NUM> of <NUM>,<NUM>-dioxane, slowly added dropwise to the amino acid solution, stirred for <NUM> at a low temperature and then transferred to a room temperature and the reaction stirred for <NUM>. after the reaction was completed, the solvent was removed through concentration by a rotary evaporator, and the residual solid was dissolved in <NUM> of water; extraction was conducted with <NUM> × <NUM> of anhydrous diethyl ether, the aqueous layer was retained and the pH was adjusted to about <NUM> with a <NUM> HCl aqueous solution to obtain a white precipitate; then extraction was conducted with <NUM> × <NUM> of ethyl acetate, the organic layer were collected and washed once with <NUM> of a saturated NaCl aqueous solution, dried with anhydrous magnesium sulfate, filtered, and then a filtrate was concentrated to obtain the white solid crude product; and the white solid obtained was further purified as follows: <NUM> of n-hexane was added, stirred for <NUM>, filtered to remove filtrate; and the solid was dried with a depressurized vacuum pump; after the obtained white solid with an appropriate amount of ethyl acetate under heating, n-hexane was added at a volume about <NUM> times for recrystallization, and stored overnight in a <NUM> refrigerator such that an increased amount of the solid product was precipitated to obtain a pure white solid, which was denoted as Fmoc-<NUM>N-Ala (C<NUM>H<NUM><NUM>NO<NUM>) and had a structure shown as follows:
<CHM>.

Fmoc-<NUM>N-Ala (C<NUM>H<NUM><NUM>NO<NUM>): <NUM>H NMR (<NUM>, CD<NUM>OD): δ= <NUM>-<NUM> (m, <NUM>), <NUM> (m, <NUM>), <NUM> (q, <NUM>), <NUM> (dt, J<NUM>= J<NUM>= <NUM>, <NUM>), <NUM> (dd, J<NUM>= J<NUM>= <NUM>, <NUM>) ppm. <NUM>C NMR (<NUM>, CD<NUM>OD): δ= <NUM>, <NUM> (d, J= <NUM>), <NUM> (d, J= <NUM>), <NUM>, <NUM>, <NUM> (d, J= <NUM>), <NUM> (d, J= <NUM>), <NUM>, <NUM>, <NUM> (d, J= <NUM>), <NUM>, <NUM> ppm. ESI-MS:[M+Na]+, m/z <NUM> (theoretical mass: <NUM>, relative error: <NUM> ppm). ESI-MS:[M-H]-, m/z <NUM> (theoretical mass: <NUM>, relative error: <NUM> ppm). Similarly, <NUM>N-D-Ala was used instead of the starting material <NUM>N-L-Ala to prepare Fmoc-<NUM>N-D-Ala (C<NUM>H<NUM><NUM>NO<NUM>) with the remaining conditions unchanged.

A double-necked flask was dried and cooled, and magnetons and weighted Fmoc-L-Ala (<NUM> mmol, namely, <NUM>) were added and dried for about <NUM>; <NUM> of oxygen-<NUM> water was prepared (<NUM>µL of acetyl chloride was added to obtain <NUM> HCl), nitrogen replacement was conducted for protection, and the flask was tightly plugged for later use; and a nitrogen balloon was prepared for later use.

A condenser tube was dried and connected to the nitrogen balloon, a dried sample and the double-necked bottle are connected to the condenser tube and the nitrogen balloon outside an infrared lamp, air in the system was replaced by a water pump, and the device was prepared and loaded with condensation water; then <NUM> of anhydrous <NUM>,<NUM>-dioxane was taken by a syringe, thoroughly stirred and then gradually heated to <NUM>; <NUM> of the prepared oxygen-<NUM> water (<NUM> mmol, namely, <NUM> equivalents) was added dropwise, and a resulting mixture was stirred to allow a reaction for <NUM> under condensation reflux. After the reaction was completed, the reaction solution was transferred to a single-necked round-bottomed flask after cooling to room temperature; and under anhydrous conditions, the same equivalent of oxygen-<NUM> water was used to allow a reaction for <NUM> under stirring and reflux, the concentration and dry were conducted to obtain a light-yellow solid powder (namely, the target compound), which was denoted as Fmoc-Ala-<NUM>OH (C<NUM>H<NUM>N<NUM>OO<NUM>) and had a structure shown as follows:
<CHM>.

Fmoc-Ala-<NUM>OH (C<NUM>H<NUM>N<NUM>OO<NUM>): <NUM>H NMR (<NUM>, CD<NUM>OD): δ= <NUM>-<NUM> (m, <NUM>), <NUM> (dd, J<NUM>= J<NUM>= <NUM>, <NUM>), <NUM> (dd, J<NUM>= J<NUM>= <NUM>, <NUM>), <NUM> (t, J= <NUM>, <NUM>), <NUM> (dt, J<NUM>= J<NUM>= <NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>) ppm. <NUM>C NMR (<NUM>, CD<NUM>OD): δ= <NUM>, <NUM>, <NUM> (d, J= <NUM>), <NUM>, <NUM>, <NUM> (d, J= <NUM>), <NUM> (d, J= <NUM>), <NUM>, <NUM>, <NUM>, <NUM>, <NUM> ppm.

ESI-MS:[M+Na]+, m/z <NUM> (theoretical mass: <NUM>, relative error: <NUM> ppm).

In order to exchange nitrogen-<NUM> of the <NUM>-fluorenylmethoxycarbonyl-L-alanine with oxygen-<NUM>, the compound Fmoc-<NUM>N-Ala (C<NUM>H<NUM><NUM>NO<NUM>) and oxygen-<NUM> water were subjected to repeated exchange reactions according to this method to obtain a target compound Fmoc-<NUM>N-Ala-<NUM>OH (C<NUM>H<NUM><NUM>N<NUM>OO<NUM>), which had a structure shown as follows:
<CHM>.

Fmoc-<NUM>N-Ala-<NUM>OH (C<NUM>H<NUM><NUM>N<NUM>OO<NUM>): <NUM>H NMR (<NUM>, CD<NUM>OD): δ= <NUM>-<NUM> (m, <NUM>), <NUM> (m, <NUM>), <NUM> (dt, J<NUM>= <NUM>, J<NUM>= <NUM>, <NUM>), <NUM> (dt, J<NUM>= J<NUM>= <NUM>, <NUM>), <NUM> (dd, J<NUM>= J<NUM>= <NUM>, <NUM>) ppm. <NUM>C NMR (<NUM>, CD<NUM>OD): δ= <NUM>, <NUM> (d, J= <NUM>), <NUM> (d, J= <NUM>), <NUM>, <NUM>, <NUM> (d, J= <NUM>), <NUM> (d, J= <NUM>), <NUM>, <NUM>, <NUM> (d, J= <NUM>), <NUM>, <NUM> ppm. ESI-MS:[M+Na]+, m/z <NUM> (theoretical mass:<NUM>, relative error: <NUM> ppm).

<NUM>-fluorenylmethoxycarbonyl-L-alanine (Fmoc-Ala, <NUM> mmol, <NUM>) and N-hydroxysuccinimide (<NUM> mmol, <NUM>) were taken and dissolved in about <NUM> of dichloromethane, cooled to about <NUM>, then DCC (<NUM> mmol, <NUM>) was added, and the reaction stirred at a low temperature for <NUM>. After the reaction was completed, the impurity DCU generated was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain the white solid crude product (<NUM>-fluorenylmethoxycarbonyl-L-alanine-N-hydroxysuccinimide (yield: <NUM>%), which would be used for the next reaction without further purification. The synthesized <NUM>-fluorenylmethoxycarbonyl-L-alanine-N-hydroxysuccinimide (<NUM> mmol) was taken and dissolved in <NUM> of acetone and <NUM> of ethanol, slowly added dropwise to a NaHCO<NUM> (<NUM> mmol, <NUM>) aqueous solution of glycine (<NUM> mmol, <NUM>) at <NUM>, subjected to a reaction at room temperature for <NUM> after the dropwise addition was completed. After the reaction was stopped, the solvent was concentrated under reduced pressure and removed, <NUM> of H<NUM>O was added to the residue, shaken for thorough mixing and then filtered to remove insoluble substances; and pH of a filtrate was adjusted with diluted hydrochloric acid to about <NUM> such that a large amount of a white solid was precipitated, filtered to obtain a gel-like solid product, and the gel-like solid product was lyophilized to obtain a white solid powder namely, the target compound Fmoc-Ala-<NUM>N-Gly (C<NUM>H<NUM>N<NUM>O<NUM>) (<NUM>,<NUM>, yield: <NUM>%), which had a structure shown as follows:
<CHM>.

Fmoc-Ala-<NUM>N-Gly (C<NUM>H<NUM>N<NUM>O<NUM>): <NUM>H NMR (<NUM>, CD<NUM>OD): δ= <NUM>-<NUM> (m, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM> (dt, J<NUM>= J<NUM>= <NUM>, <NUM>), <NUM> (q, J= <NUM>, <NUM>), <NUM> (dt, J<NUM>= J<NUM>= <NUM>, <NUM>), <NUM> (d, J= <NUM>, <NUM>) ppm. <NUM>C NMR (<NUM>, CD<NUM>OD): δ= <NUM> (d, J= <NUM> ), <NUM>, <NUM>, <NUM> (d, J= <NUM>), <NUM>, <NUM>, <NUM>, <NUM> (d, J= <NUM>), <NUM>, <NUM>, <NUM> (d, J= <NUM>), <NUM>, <NUM> (d, J= <NUM>), <NUM> ppm. ESI-MS: [M+Na]+, m/z <NUM> (theoretical mass: <NUM>, relative error: <NUM> ppm).

Similarly, in order to synthesize a <NUM>-fluorenylmethoxycarbonyl-L-alanine-glycine dipeptide labeled with other stable isotopes according to the above method, <NUM>-fluorenylmethoxycarbonyl-L/D-alanine labeled with different isotopes was first subjected to a reaction with N-hydroxysuccinimide to obtain an activated ester, and then the activated ester was subjected to a reaction with glycine labeled with different isotopes to obtain the following target compounds Fmoc-<NUM>N-Ala-Gly (C<NUM>H<NUM>N<NUM>O<NUM>), which had a structure shown as follows:
<CHM>.

Fmoc-<NUM>N-Ala-Gly (C<NUM>H<NUM>N<NUM>O<NUM>): <NUM>H NMR (<NUM>, CD<NUM>OD): δ= <NUM>-<NUM> (m, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM> (m, <NUM>), <NUM> (t, J= <NUM>, <NUM>), <NUM> (dt, J<NUM>= J<NUM>= <NUM>, <NUM>), <NUM> (dd, J= <NUM>, <NUM>) ppm. <NUM>C NMR (<NUM>, CD<NUM>OD): δ=<NUM>, <NUM>, <NUM>(d, J= <NUM>), <NUM> (d, J= <NUM>), <NUM>, <NUM>, <NUM>(d, J= <NUM>), <NUM> (d, J= <NUM>), <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> ppm.

ESI-MS: [M+Na]+, m/z <NUM> (theoretical mass: <NUM>, relative error: <NUM> ppm).

Fmoc-Ala-<NUM>OH-<NUM>N-Gly (C<NUM>H<NUM>N<NUM>O<NUM>), which had a structure shown as follows:
<CHM>.

Fmoc-Ala-<NUM>OH-<NUM>N-Gly (C<NUM>H<NUM>N<NUM>O<NUM>): <NUM>H NMR (<NUM>, CD<NUM>OD): δ= <NUM>-<NUM> (m, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM> (dt, J<NUM>= <NUM>, J<NUM>= <NUM>, <NUM>), <NUM> (q, J= <NUM>, <NUM>), <NUM> (dt, J<NUM>= J<NUM>= <NUM>, <NUM>), <NUM> (d, J= <NUM>,<NUM>) ppm. <NUM>C NMR (<NUM>, CD<NUM>OD): δ= <NUM> (d, J= <NUM>), <NUM>, <NUM>, <NUM> (d, J= <NUM>), <NUM>, <NUM>, <NUM> (d, J= <NUM>), <NUM> (d, J= <NUM>), <NUM>, <NUM>, <NUM> (d, J= <NUM>), <NUM>, <NUM> (d, J= <NUM>), <NUM> ppm.

Fmoc-Ala-<NUM>OH-<NUM>N,<NUM>C<NUM>-Gly (C<NUM>H<NUM>N<NUM>O<NUM>), which had a structure shown as follows:
<CHM>.

Fmoc-Ala-<NUM>OH-<NUM>N,<NUM>C<NUM>-Gly (C<NUM>H<NUM>N<NUM>O<NUM>): <NUM>H NMR (<NUM>, CD<NUM>OD): δ= <NUM>-<NUM> (m, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM> (m, <NUM>), <NUM> (dd, J<NUM>= J<NUM>= <NUM>, <NUM>), <NUM> (dd, J<NUM>= J<NUM>= <NUM>, <NUM>), <NUM> (dt, J<NUM>= J<NUM>= <NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>) ppm. <NUM>C NMR (<NUM>, CD<NUM>OD): δ= <NUM>, <NUM> (d, J= <NUM>), <NUM>, <NUM> (d, J= <NUM>), <NUM>, <NUM>, <NUM> (d, J= <NUM>), <NUM> (d, J= <NUM>), <NUM>, <NUM>, <NUM> (d, J= <NUM>), <NUM>, <NUM> (dt, J= <NUM>), <NUM> ppm.

Fmoc-<NUM>N-Ala-<NUM>OH-Gly (C<NUM>H<NUM>N<NUM>O<NUM>), which had a structure shown as follows:
<CHM>.

Fmoc-<NUM>N-Ala-<NUM>OH-Gly (C<NUM>H<NUM>N<NUM>O<NUM>): <NUM>H NMR (<NUM>, CD<NUM>OD): δ= <NUM>-<NUM> (m, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM> (dd, J<NUM>= <NUM>, J<NUM>= <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (dt, J<NUM>= J<NUM>= <NUM>, <NUM>), <NUM> (dd, J<NUM>= J<NUM>= <NUM>, <NUM>) ppm. <NUM>C NMR (<NUM>, CD<NUM>OD): δ= <NUM>, <NUM>, <NUM>, <NUM> (d, J= <NUM>), <NUM>, <NUM>, <NUM> (d, J= <NUM>), <NUM> (d, J= <NUM>), <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> ppm.

Fmoc-<NUM>N-Ala-<NUM>OH-<NUM>C<NUM>-Gly (C<NUM>H<NUM>N<NUM>O<NUM>), which had a structure shown as follows:
<CHM>.

Fmoc-<NUM>N-Ala-<NUM>OH-<NUM>C<NUM>-Gly (C<NUM>H<NUM>N<NUM>O<NUM>): <NUM>H NMR (<NUM>, CD<NUM>OD): δ= <NUM>-<NUM> (m, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM> (dt, J<NUM>= J<NUM>= <NUM>, <NUM>), <NUM> (t, J= <NUM>, <NUM>), <NUM> (t, J= <NUM>, <NUM>), <NUM> (dt, J<NUM>= J<NUM>= <NUM>, <NUM>), <NUM> (dd, J= <NUM>, <NUM>) ppm. <NUM>C NMR (<NUM>, CD<NUM>OD): δ= <NUM>, <NUM> (d, J= <NUM>), <NUM>, <NUM> (d, J= <NUM>), <NUM>, <NUM>, <NUM> (d, J= <NUM>), <NUM> (d, J= <NUM>), <NUM>, <NUM>, <NUM>, <NUM>, <NUM> (dd, J= <NUM>), <NUM> ppm.

The <NUM>-fluorenylmethoxycarbonyl-alanine-glycine dipeptide (<NUM> mmol) labeled with stable isotopes prepared in the step <NUM> was taken and dissolved in <NUM> of methanol, two drops of glacial acetic acid were added, and one-tenth of the mass of <NUM>% palladium on carbon was added; after the reaction reagents were added, air was removed by a vacuum pump, then an inert gas was introduced and then replaced with hydrogen, and the reaction was stirred overnight at room temperature. When it was determined by a thin layer chromatography spotting plate that the reaction was completed, the palladium on carbon was removed through filtration with a filter aid of diatomite, and the solvent was removed by a rotary evaporator; and about <NUM> of distilled water was added to a residue to dissolve, extraction was conducted with <NUM> of each of trichloromethane and ethyl acetate, and the aqueous layer were retained, lyophilized, and purified through HPLC to obtain a stable isotope-labeled alanine-glycine dipeptide <NUM>N-Ala-Gly (C<NUM>H<NUM>N<NUM>O<NUM>), which had a structure shown as follows:
<CHM>.

<NUM>N-Ala-Gly (C<NUM>H<NUM>N<NUM>O<NUM>): <NUM>H NMR (<NUM>, D<NUM>O): δ= <NUM> (q, J= <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (dd, J<NUM>= J<NUM>= <NUM>, <NUM>) ppm.

ESI-MS: [M-H]-, m/z <NUM> (theoretical mass: <NUM>, relative error: <NUM> ppm).

Similarly, with reference to the same method, the <NUM>-fluorenylmethoxycarbonyl-alanine-glycine dipeptide compounds labeled with other stable isotopes prepared in step <NUM> were subjected to a de-Fmoc treatment to obtain the following compounds:.

ESI-MS: [M-H]-,m/z <NUM> (theoretical mass: <NUM>, relative error: <NUM> ppm).

Ala-<NUM>OH-<NUM>N-Gly (C<NUM>H<NUM>N<NUM>O<NUM>), which had a structure shown as follows:
<CHM>.

Ala-<NUM>OH-<NUM>N-Gly (C<NUM>H<NUM>N<NUM>O<NUM>): <NUM>H NMR (<NUM>, D<NUM>O): δ= <NUM> (q, J= <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J= <NUM>, <NUM>) ppm.

Ala-<NUM>OH-<NUM>N,<NUM>C<NUM>-Gly (C<NUM>H<NUM>N<NUM>O<NUM>), which had a structure shown as follows:
<CHM>.

Ala-<NUM>OH-<NUM>N,<NUM>C<NUM>-Gly (C<NUM>H<NUM>N<NUM>O<NUM>): <NUM>H NMR (<NUM>, D<NUM>O): δ= <NUM> (q, J= <NUM>, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM> (d, J= <NUM>, <NUM>) ppm.

ESI-MS: [M-H]-, m/z <NUM> (theoretical mass: <NUM>, relative error: <NUM> ppm).

<NUM>N-Ala-<NUM>OH-Gly (C<NUM>H<NUM>N<NUM>O<NUM>), which had a structure shown as follows:
<CHM>.

<NUM>N-Ala-<NUM>OH-Gly (C<NUM>H<NUM>N<NUM>O<NUM>): <NUM>H NMR (<NUM>, D<NUM>O): δ= <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (dd, J<NUM>= J<NUM>= <NUM>, <NUM>) ppm.

ESI-MS: [M-H]-, m/z <NUM> (theoretical mass:<NUM>, relative error:<NUM> ppm).

<NUM>N-Ala-<NUM>OH-<NUM>C<NUM>-Gly (C<NUM>H<NUM>N<NUM>O<NUM>), which had a structure shown as follows:
<CHM>.

<NUM>N-Ala-<NUM>OH-<NUM>C<NUM>-Gly (C<NUM>H<NUM>N<NUM>O<NUM>): <NUM>H NMR (<NUM>, D<NUM>O): δ= <NUM> (m, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM> (dd, J<NUM>= J<NUM>= <NUM>, <NUM>) ppm.

Absolute ethanol (<NUM> mmol, <NUM>, <NUM>) was taken and dissolved in <NUM> of dichloromethane to obtain an ethanol solution; phosphorus trichloride (<NUM> mmol, <NUM>, <NUM>µL) was dissolved in <NUM> of dichloromethane to obtain a phosphorus trichloride solution, and the phosphorus trichloride solution was cooled to <NUM>; under the protection of nitrogen, the ethanol solution was slowly added dropwise to the phosphorus trichloride solution. The reaction system was transferred to room temperature and the reaction stirred, during which a phosphorus spectrum was tracked every half an hour; <NUM> to <NUM> later, the reaction was completed, at which point the phosphorus trichloride was exhausted. The organic solvent and hydrochloric acid gas were removed through concentration under reduced pressure by a rotary evaporator. Distillation was conducted under reduced pressure to obtain a colorless or light-yellow oily liquid, which was diethyl phosphite. Similarly, in order to synthesize diethyl phosphite labeled with the stable isotope carbon-<NUM>, the raw material ethanol was replaced by ethanol labeled with deuterium-<NUM> and carbon-<NUM> to obtain a series of diethyl phosphites labeled with deuterium-<NUM> and carbon-<NUM>, which were as follows:.

ESI-MS: [M+H]+, m/z <NUM> (theoretical mass: <NUM>, relative error: <NUM> ppm).

<NUM>C<NUM>-DEPH (<NUM>C<NUM>H<NUM>O<NUM>P), which had a structure shown as follows:
<CHM>.

<NUM>C<NUM>-DEPH (<NUM>C<NUM>H<NUM>O<NUM>P): <NUM>H NMR (<NUM>, CDCl<NUM>): δ= <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>) ppm. <NUM>C NMR (<NUM>, CDCl<NUM>): δ= <NUM> (dd, J<NUM>= <NUM>, J<NUM>= <NUM>), <NUM> (dd, J<NUM>= J<NUM>=<NUM>) ppm. <NUM>P NMR (<NUM>, CDCl<NUM>): δ= <NUM> ppm.

D<NUM><NUM>C<NUM>-DEPH (<NUM>C<NUM> C<NUM> D<NUM>H<NUM>O<NUM>P), which had a structure shown as follows:
<CHM>.

D<NUM><NUM>C<NUM>-DEPH (<NUM>C<NUM> C<NUM> D<NUM>H<NUM>O<NUM>P): <NUM>H NMR (<NUM>, CDCl<NUM>): δ= <NUM> (s, <NUM>), <NUM>(s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>) ppm. <NUM>C NMR (<NUM>, CDCl<NUM>): δ= <NUM>-<NUM> (m), <NUM> (dd, J<NUM>= <NUM>, J<NUM>= <NUM>) ppm. <NUM>P NMR (<NUM>, CDCl<NUM>): δ= <NUM> ppm.

The reaction conditions were similar to that in Example <NUM>, except that the stable isotope-labeled alanine-glycine dipeptide obtained in the step <NUM> was used instead of the ordinary alanine-glycine dipeptide and the carbon-<NUM>-labeled diethyl phosphite obtained in the step <NUM> was used instead of the ordinary diethyl phosphite to prepare a stable isotope-labeled diethyl phosphite-alanine-glycine dipeptide, and finally, the stable isotope-labeled diethyl phosphite-alanine-glycine dipeptide was subjected to activation with N-hydroxysuccinimide to prepare the following six-labeled reagents with isobaric stable isotopes.

DEPH-Ala-<NUM>OH-<NUM>N,<NUM>C<NUM>-Gly (C<NUM>H<NUM>N<NUM>O<NUM>P), which had a structure shown as follows:
<CHM>.

DEPH-Ala-<NUM>OH-<NUM>N,<NUM>C<NUM>-Gly (C<NUM>H<NUM>N<NUM>O<NUM>P): <NUM>H NMR (<NUM>, CDCl<NUM>): δ= <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (m, <NUM>), <NUM> (qd, J<NUM>= J<NUM>= <NUM>, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM> (m, <NUM>) ppm. <NUM>C NMR (<NUM>, CDCl<NUM>): δ= <NUM> (d, J= <NUM>), <NUM>, <NUM> (dd, J<NUM>=<NUM>, J<NUM>=<NUM>), <NUM>, <NUM> (dd, J<NUM>= J<NUM>= <NUM>), <NUM>, <NUM> (dd, J<NUM>= J<NUM>= <NUM>) ppm. <NUM>P NMR (<NUM>, CDCl<NUM>): δ= <NUM> ppm.

DEPH-<NUM>N-Ala-<NUM>OH-<NUM>C<NUM>-Gly, which had a structure shown as follows:
<CHM>.

DEPH-<NUM>N-Ala-<NUM>OH-<NUM>C<NUM>-Gly (C<NUM>H<NUM>N<NUM>O<NUM>P): <NUM>H NMR (<NUM>, CDCl<NUM>): δ= <NUM> (s, <NUM>), <NUM> (q, J= <NUM>, <NUM>), <NUM> (qd, J<NUM>= J<NUM>= <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>),<NUM> (d, J= <NUM>, <NUM>), <NUM> (t, J= <NUM>, <NUM>) ppm. <NUM>C NMR (<NUM>, CDCl<NUM>): δ= <NUM> (d, J= <NUM>), <NUM>, <NUM>, <NUM>, <NUM> (d, J= <NUM>), <NUM>, <NUM> ppm. <NUM>P NMR (<NUM>, CDCl<NUM>): δ= <NUM> (d, J= <NUM>) ppm.

<NUM>C<NUM>-DEPH-Ala-<NUM>OH-<NUM>N-Gly (C<NUM>H<NUM>N<NUM>O<NUM>P), which had a structure shown as follows:
<CHM>.

<NUM>C<NUM>-DEPH-Ala-<NUM>OH-<NUM>N-Gly (C<NUM>H<NUM>N<NUM>O<NUM>P): <NUM>H NMR (<NUM>, CDCl<NUM>): δ= <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>), <NUM> (qd, J<NUM>= J<NUM>= <NUM>, <NUM>), <NUM> (dt, J<NUM>= J<NUM>= <NUM>, <NUM>), <NUM> (m, <NUM>) ppm. <NUM>C NMR (<NUM>, CDCl<NUM>): δ= <NUM>, <NUM>, <NUM> (dd, J<NUM>= <NUM>, J<NUM>= <NUM>), <NUM>, <NUM> (d, J= <NUM>), <NUM>, <NUM> (dd, J<NUM>= J<NUM>= <NUM>) ppm. <NUM>P NMR (<NUM>, CDCl<NUM>): δ= <NUM> ppm.

<NUM>C<NUM>-DEPH-<NUM>N-Ala-<NUM>OH-Gly (C<NUM>H<NUM>N<NUM>O<NUM>P), which had a structure shown as follows:
<CHM>.

<NUM>C<NUM>-DEPH-<NUM>N-Ala-<NUM>OH-Gly (C<NUM>H<NUM>N<NUM>O<NUM>P): <NUM>H NMR (<NUM>, CDCl<NUM>): δ= <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J= <NUM>, <NUM>),<NUM> (m, <NUM>), <NUM> (qd, J= <NUM>, <NUM>), <NUM> (t, J= <NUM>, <NUM>), <NUM> (m, <NUM>) ppm. <NUM>C NMR (<NUM>, CDCl<NUM>): δ= <NUM> (d, J= <NUM>), <NUM>, <NUM> (dd, J<NUM>= <NUM>, J<NUM>= <NUM>), <NUM>, <NUM>, <NUM> (d, J= <NUM>), <NUM> (dd, J<NUM>= J<NUM>= <NUM>) ppm. <NUM>P NMR (<NUM>, CDCl<NUM>): δ= <NUM> (dt, J<NUM>= J<NUM>= <NUM>) ppm.

<NUM>C<NUM>-DEPH-Ala-<NUM>N-Gly (C<NUM>H<NUM>N<NUM>O<NUM>P), which had a structure shown as follows:
<CHM>.

<NUM>C<NUM>-DEPH-Ala-<NUM>N-Gly (C<NUM>H<NUM>N<NUM>O<NUM>P): <NUM>H NMR (<NUM>, CDCl<NUM>): δ= <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>), <NUM> (qd, J<NUM>= J<NUM>= <NUM>, <NUM>), <NUM> (t, J= <NUM>, <NUM>), <NUM> (m, <NUM>) ppm. <NUM>C NMR (<NUM>, CDCl<NUM>): δ= <NUM> (dd, J<NUM>= J<NUM>= <NUM>), <NUM>, <NUM> (dd, J<NUM>= J<NUM>= <NUM>), <NUM>, <NUM> (d, J= <NUM>), <NUM> (d, J= <NUM>), <NUM> (m) ppm. <NUM>P NMR (<NUM>, CDCl<NUM>): δ= <NUM> ppm.

<NUM>C<NUM>-DEPH-<NUM>N-Ala-Gly (C<NUM>H<NUM>N<NUM>O<NUM>P), which had a structure shown as follows:
<CHM>.

<NUM>C<NUM>-DEPH-<NUM>N-Ala-Gly (C<NUM>H<NUM>N<NUM>O<NUM>P): <NUM>H NMR (<NUM>, CDCl<NUM>): δ= <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>), <NUM>(t, J= <NUM>, <NUM>), <NUM> (m, <NUM>) ppm. <NUM>C NMR (<NUM>, CDCl<NUM>): δ= <NUM> (d, J= <NUM>), <NUM>, <NUM> (dd, J= <NUM>), <NUM>, <NUM>, <NUM> (d, J= <NUM>), <NUM> (m) ppm. <NUM>P NMR (<NUM>, CDCl<NUM>): δ= <NUM> ppm.

D<NUM><NUM> C<NUM>-DEPH-<NUM>N-Ala-Gly (C<NUM>H<NUM>N<NUM>O<NUM>P), which had a structure shown as follows:
<CHM>.

D<NUM><NUM>C<NUM>-DEPH-i5N-Ala-Gly (C<NUM>H<NUM>N<NUM>O<NUM>P): <NUM>H NMR (<NUM>, CDCl<NUM>): δ= <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>), <NUM>(t, J= <NUM>, <NUM>), <NUM> (m, <NUM>) ppm. <NUM>C NMR (<NUM>, CDCl<NUM>): δ= <NUM> (d, J= <NUM>), <NUM>, <NUM>(dd, J= <NUM>), <NUM>, <NUM>, <NUM> (d, J= <NUM>), <NUM> (m) ppm. <NUM>P NMR (<NUM>, CDCl<NUM>): δ= <NUM> ppm.

Example <NUM> Total proteolytic peptides extracted from Hela cells and derivatization analysis with an iSIPL<NUM> reagent.

Taking the extraction of total proteolytic peptides from Hela cells as an example, the medium used for the culture was DMEM medium, and the cell culture conditions were <NUM> and <NUM>% CO<NUM>.

The cellular protein was extracted through the following steps:.

Derivatization reaction steps: A concentration of peptides obtained after enzymolysis was determined by a protein concentration detector, and a peptide solution was evenly dispensed into several tubes (<NUM>µg to <NUM>µg per tube). Two of the tubes were taken to conduct an experiment, with one tube as a blank control and the other tube for a derivatization reaction with an iSIPL<NUM> reagent. iSIPL<NUM> was dissolved in <NUM>µL of acetonitrile, added to <NUM>µL of a peptide solution, thoroughly shaken, subjected to a reaction in an ice bath for half an hour, and then transferred to room temperature and subjected to a reaction for half an hour; and the acetonitrile was removed through dry centrifugation, and subjected to acid adjustment, desalination, and MS analysis.

A structure of the iSIPL<NUM> labeling reagent was as follows:
<CHM>
LC-MS/MS analysis.

LC-MS/MS analysis conditions were as follows: nano-UHPLC Proxeon Easy-nLC <NUM> coupled LTQ-Orbitrap Q-Exactive MS of Thermo Scientific was adopted; after being injected into a liquid chromatograph, a sample was first captured by a capture column (C18, <NUM>, <NUM> × <NUM>), then entered an analytical column (Acclaim PepMap RSLC-C18, <NUM>, <NUM> × <NUM>µ m), and was separated, with a total injection amount of <NUM>µg of polypeptides. Chromatographic conditions: a gradient elution was conducted for <NUM> with <NUM>% to <NUM>% of acetonitrile with <NUM>% formic acid; flow rate: <NUM> nL/min. An effluent from the column directly entered an electrospray ionization source of the MS. HCD fragments were used for a Q-exactive platform. The top <NUM> ions in abundance were selected to allow crushing and dynamic exclusion for <NUM>. The original data files were generated by Xcalibur software (Thermo Scientific) and processed by Proteome Discoverer V2. <NUM>; and the human protein database of Swiss-Prot was searched by the search software SEQUEST to complete the peptide identification and quantitative analysis. The results of protein identification were shown in Table <NUM>:.

It can be seen from Table <NUM> that, when the total proteolytic peptides extracted from Hela cells were only used as a blank control without undergoing a derivatization reaction, <NUM>,<NUM> proteins were searched; and when the total proteolytic peptides were subjected to a derivatization reaction with a iSIPL<NUM> labeling reagent, <NUM>,<NUM> proteins were searched, indicating that the labeling reagent exhibited high labeling efficiency with low protein loss.

Example <NUM> Comparative analysis of a labeling reagent iSIPL<NUM> and a commercial labeling reagent TMT<NUM>
The experimental methods of total protein extraction and enzymolysis to produce peptides were the same as that in Example <NUM>.

A concentration of the peptides obtained after enzymolysis was determined, and a peptide solution was evenly dispensed into four tubes for test. <NUM>µL of a solution of iSIPL<NUM> (<NUM>) in acetonitrile was added to each of two of the tubes with a ratio of m(iSIPL<NUM>) to m(peptide) being <NUM>:<NUM>, such that two parallel test tubes were set; and <NUM>µL of a solution of TMT<NUM> (<NUM>) in acetonitrile was added to each of the other two tubes with a ratio of m(TMT0) to m(peptide) being <NUM>:<NUM>, such that two parallel test tubes were set. After the derivatization reaction was completed, the acid adjustment, desalination, and MS analysis were conducted. A fragment ion peak of mass spectrometry analysis was shown in <FIG>; and protein identification results were shown in Table <NUM> and <FIG>.

It can be seen from Table <NUM> and <FIG> that, for <NUM>µ g of labeled Hela peptides, <NUM>,<NUM> proteins were detected with iSIPL<NUM> and <NUM>,<NUM> proteins were detected with TMT<NUM>, indicating that the iSIPL labeling reagent exhibited a prominent identification effect and high detection repeatability for trace proteins.

Example <NUM> BSA standard peptide concentration gradient experiment: different concentrations of standard peptides were subjected to derivatization with iSIPL<NUM>.

This Example was the same as Example <NUM> except that, a BSA standard protein was used instead of the total protein extracted from Hela cells for reductive alkylation and enzymolysis to produce peptides; the peptides obtained after enzymolysis of the BSA standard protein were tested for concentration and diluted to different concentrations of <NUM>λ, <NUM>λ, <NUM>λ, <NUM>λ, <NUM>λ, and the like to conduct a standard peptide concentration gradient experiment. The different concentrations of BSA standard peptides were each subjected to derivatization with the iSIPLzero reagent at a same mass to determine the lowest concentration of peptides that could be identified by the chemical labeling reagent.

The detection results were shown in Table <NUM>.

Standard proteolysis: <NUM>µg of a BSA protein was dissolved in a <NUM> Tris/HCl pH <NUM> (<NUM> urea) buffer, added to a <NUM> KD concentration tube and centrifuged at <NUM>,<NUM> until about <NUM>µL was left; <NUM>µL of a DTT solution was added, thoroughly mixed, allowed to stand at room temperature for <NUM>, and centrifuged at <NUM>,<NUM> for <NUM>; <NUM>µL of an IAA solution was added, thoroughly mixed, allowed to stand at room temperature for <NUM>, centrifuged at <NUM>,<NUM> for <NUM>; <NUM>µL of a <NUM> Tris/HCl pH <NUM> (<NUM> urea) solution was added to the concentration tube and the concentration tube was centrifuged at <NUM>,<NUM> for <NUM>, and the operation was repeated twice; <NUM> uL of a triethylammonium bicarbonate solution with trypsin (a ratio of the trypsin to the protein was <NUM>:<NUM>) was added to the concentration tube, thoroughly mixed and incubated at <NUM> for <NUM>; a collection tube was changed, and the concentration tube was centrifuged at <NUM>,<NUM> for <NUM>; <NUM>µL of <NUM> NaCl was added, and the concentration tube was centrifuged at <NUM>,<NUM> for <NUM>; and a resulting product was acidified with formic acid (with a pH of <NUM> to <NUM>) and subjected to desalination according to the desalination method in Example <NUM> and then to MS analysis.

Example <NUM> Concentration gradient experiment of total proteolytic peptides extracted from Hela cells: different concentrations of peptides were each subjected to derivatization with iSIPL<NUM>.

Like Example <NUM>, the total protein was extracted from Hela cells and subjected to reductive alkylation, enzymolysis, and then derivatization to obtain a proteolysis solution with a total protein concentration of <NUM>µ g/µL, and the proteolysis solution was diluted to the different concentrations of <NUM>µg/µL, <NUM>µg/µL, <NUM>µg/µL, <NUM>µg/µL, <NUM>µg/µL, and the like to conduct an enzymatic peptide concentration gradient experiment. The different concentrations of peptides extracted from HELA cells (<NUM>µL) were subjected to derivatization with the iSIPLzero reagent at a same mass (<NUM>), and after the reaction was completed, a resulting product was desalted according to Example <NUM>, detected by MS, and searched in a protein library.

The test results were shown in Table <NUM>.

Two samples (<NUM>µg of the Hela total proteolytic peptides) were taken; iSIPL<NUM>-<NUM> was added to one of the two samples and an equal amount of iSIPL<NUM>-<NUM> was added to the other one, a derivatization reaction was conducted at a low temperature for half an hour and then at room temperature for <NUM>. After the reaction was completed, the acetonitrile was removed by centrifugal drying, a pH of a residue was adjusted to <NUM> to <NUM> with <NUM>% to <NUM>% formic acid, and a resulting product was then desalted according to Example <NUM> and tested by MS. MS data was analyzed by the MaxQuant software. The results showed that a total of <NUM>,<NUM> proteins were detected, among which <NUM>,<NUM> proteins had a report ion strength ratio of <NUM> to <NUM>, indicating that the iSIPL<NUM>-plex labeling reagent could well quantify a complex protein sample.

Structures of the two iSIPL labeling reagents were as follows:.

A specified amount of the BSA standard protein was weighed, subjected to reductive alkylation and enzymolysis, tested for concentration, and divided into six portions according to a mass ratio of <NUM>:<NUM>:<NUM>: <NUM>:<NUM>: <NUM>. The six labeling reagents of iSIPL<NUM>-<NUM>, iSIPL<NUM>-<NUM>, iSIPL<NUM>-<NUM>, iSIPL<NUM>-<NUM>, iSIPL<NUM>-<NUM>, and iSIPL<NUM>-<NUM> were added at an equal amount to the six portions, respectively, and a derivatization reaction was conducted at a low temperature for half an hour and then at room temperature for <NUM>. After the reaction was completed, the acetonitrile was removed by centrifugal drying, a pH of a residue was adjusted to <NUM> to <NUM> with <NUM>% to <NUM>% formic acid, and a resulting product was then desalted according to Example <NUM>, tested by MS, and searched in a protein library. Some peptides were selected for sequence analysis, as shown in Table <NUM>.

It can be seen from Table <NUM> that there is an obvious characteristic reporter ion peak m/z <NUM>-<NUM> in the reporter ion generation region, indicating that the phosphate group has high sensitivity in MS detection, and is successfully labeled with the six labeling reagents. The quantification result is accurate.

Structures of the iSIPL<NUM> labeling reagents were as follows:.

A protein was extracted from a urine sample by a method with reference to a literature. <NUM>µL of urine was taken and centrifuged, the supernatant was added to a filter (<NUM>) and centrifuged at <NUM>,<NUM> rpm, and the protein in the centrifuge tube was subjected to reductive alkylation with reference to Example <NUM>, tested for concentration, and divided into six portions; the six labeling reagents of iSIPL<NUM>-<NUM>, iSIPL<NUM>-<NUM>, iSIPL<NUM>-<NUM>, iSIPL<NUM>-<NUM>, iSIPL<NUM>-<NUM>, and iSIPL<NUM>-<NUM> were added at an equal amount to the six portions, respectively; a derivatization reaction was conducted at a low temperature for half an hour and then at room temperature for <NUM>; and after the reaction was completed, the acetonitrile was removed by centrifugal drying, a pH of a residue was adjusted to <NUM> to <NUM> with <NUM>% to <NUM>% formic acid, and a resulting product was then desalted according to Example <NUM> and tested by MS. MS data was analyzed by the MaxQuant software. The results showed that a total of <NUM>,<NUM> proteins were detected, among which <NUM>,<NUM> proteins had a report ion strength ratio of <NUM> to <NUM>, indicating that the iSIPL<NUM> labeling reagent could well quantify a protein in a urine sample.

Claim 1:
An isobaric stable isotope-containing phosphorylated protein labeling reagent comprising a structural formula as follows:
<CHM>
wherein a is <NUM> or <NUM>; b is <NUM> or <NUM>; c is <NUM> or <NUM>; d is <NUM> or <NUM>; n is <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> and preferably <NUM> or <NUM>; n<NUM> is <NUM>, <NUM>, or <NUM> and preferably <NUM> or <NUM>; R<NUM> is a side chain substituent of an L or D-configuration amino acid with or without a stable isotope, and preferably R<NUM> is methyl; and R<NUM> is a side chain substituent of an amino acid with or without a stable isotope, and preferably R<NUM> is hydrogen.