Method for target peptide quantification based on mass spectrometry using internal standard peptides labeled with stable isotope

Abstract

The present invention relates to a method for target peptide quantification based on mass spectrometry using internal standard peptides labeled with a stable isotope, in which, for quantification of a target peptide with a known sequence, standard peptides having the same sequence as the target peptide and including various amino acids substituted with an isotope are synthesized, and then mixed with the target peptide at various concentrations, and through LC-MS analysis of the mixtures, an internal standard curve is generated and, at the same time, the target (endogenous) peptide is analyzed, by single analysis of the mixed samples, and the amount of the target (endogenous) peptide is accurately quantified from each of the peak areas obtained through the analysis.

Description

Mass spectrometry-based quantitative method for target peptides using stable isotope-labeled internal standard peptides

[0001] The present invention relates to a mass spectrometry-based method for quantifying a target peptide using a stable isotope-labeled internal standard peptide.

[0002] This work was supported by the following projects: "Discovering Immuno-Oncology Therapeutic Targets through Structural Proteomic Analysis of Multi-Tumor Single-Cells" (Project No. 1711194258, 00209456) funded by the Ministry of Science and ICT; "Discovering New Liver Cancer Therapeutic Targets Based on Structural Proteomic Data" (Project No. 1711172806, 2022R1F1A1074235) funded by the Ministry of Science and ICT; and "Fostering Artificial Intelligence Convergence Innovation Talent" (Project No. 1711179294, RS-2022-00155857) funded by the Ministry of Science and ICT.

[0003] Mass spectrometry of a polypeptide mixture can identify or quantify proteins in the mixture by interpreting the mass spectrum. Peaks in the mass spectrum data are defined by the mass-to-charge ratio (m / z) and intensity of the polypeptides in the mixture, and the polypeptide mixture is analyzed by mass spectrometry equipment using protons H. + They become ions with a positive (+) charge when attached, and instead of their direct mass, polypeptide ions are detected in the mass spectrum by their mass-to-charge ratio (m / z), which is the mass divided by the charge, and the intensity of the corresponding ion. By predefining the mass-to-charge ratio (m / z) and the mass-to-charge ratio (m / z) of the fragmented peptide, the target polypeptide can be quantified through mass analysis.

[0004] The mass of a polypeptide is defined as the sum of the masses of, for example, carbon (C), hydrogen (H), nitrogen (N), oxygen (O), and sulfur (S) atoms that make up the polypeptide, and the monoisotopic mass is used as a representative value. The monoisotopic mass refers to the sum of the masses of the atoms that make up the polypeptide assuming that all the atoms are composed of the lightest isotope. All elements existing in nature have isotopes, for example, even the same carbon atom 12 C, 13 C isotope exists 13 In the case of C, it has an abundance ratio of 1%. Therefore, even in the same polypeptide, if some atoms correspond to heavy isotopes, multiple peaks with different mass values ​​can be detected in the spectrum. For this reason, the monoisotopic mass is used as a value representing the mass of the polypeptide. However, in an actual spectrum, peaks due to the difference in isotopic mass of several polypeptides can appear in a complex overlapping manner, and as the mass of the polypeptide increases, the probability that all of the constituent atoms are composed of the lightest isotope decreases, making it difficult to directly find the peak corresponding to the monoisotopic mass in the spectrum.

[0005] Meanwhile, Korean Patent Registration No. 0789430 discloses a method and a recording medium for determining a monoisotopic mass by finding an isotope group of a polypeptide from a mass spectrum of a complex polypeptide mixture, and Korean Patent Publication No. 2023-0135370 discloses a method for isotope labeling of a glycoprotein using transglycosylation of an endoglycosidase and an isotope-labeled amino sugar oxazoline for quantitative mass spectrometry. However, a mass spectrometry-based method for quantifying a target peptide using a stable isotope-labeled internal standard peptide of the present invention has not yet been disclosed.

[0006] The present invention was derived from the above-mentioned needs, and the present invention provides a mass spectrometry-based method for quantifying a target peptide using an internal standard peptide labeled with a stable isotope, and, when quantifying a target peptide of which the sequence is known, a standard peptide having the same sequence as the target peptide and including various amino acids substituted with isotopes is synthesized, and then mixed with the target peptide at various concentrations, and a method for quantifying the amount of the target (endogenous) peptide from each peak area obtained through liquid chromatography-mass spectrometry (LC-MS) analysis of the mixture is established, thereby completing the present invention.

[0007] In order to achieve the above purpose, the present invention comprises: (1) a step of synthesizing multiple types of internal standard peptides having the same amino acid sequence as the target peptide, in which amino acids at both terminals or one terminal are substituted with stable isotopes, and in which the number of substituted amino acids is n or less when the number of amino acids of the target peptide to be quantified is n;

[0008] (2) A step of adding the internal standard peptide synthesized in step (1) to a sample containing the target peptide to be quantified, and mixing the amount of the internal standard peptide added in a proportional or inversely proportional amount according to the number of amino acids containing the substituted stable isotope;

[0009] (3) After the above step (2), a step of performing LC (Liquid Chromatography)-MS (Mass Spectrometry) or LC-MS / MS analysis on a sample containing the internal standard peptide and target peptide;

[0010] (4) A step of obtaining the peak area value of the target peptide obtained from the LC-MS analysis performed in the above step (3); and the peak area value of each internal standard peptide substituted with a stable isotope having a different amount;

[0011] (5) A step of obtaining an internal standard curve (Internal Standard Curve, Calibration Curve) formula from the area values ​​of internal standard peptides having different amounts obtained in the above step (4); and

[0012] (6) A method for quantifying a target peptide based on mass spectrometry using a stable isotope-labeled internal standard peptide, including a step of quantifying by substituting the area value of the target peptide obtained in step (4) into the internal standard curve formula obtained in step (5).

[0013] In addition, the present invention comprises (1) a step of synthesizing multiple types of internal standard peptides having the same sequence as the target peptide, in which amino acids at both terminals or one terminal are substituted with stable isotopes, and in which the number of substituted amino acids is n or less when the number of amino acids of the target peptide to be determined is n;

[0014] (2) A step of adding the internal standard peptide synthesized in step (1) to a sample containing the target peptide to be determined, and mixing the internal standard peptide by adding an amount of the internal standard peptide that is proportional or inversely proportional to the number of amino acids containing the substituted stable isotope;

[0015] (3) After the above step (2), a step of performing LC (Liquid Chromatography)-MS (Mass Spectrometry) or LC-MS / MS analysis on a sample containing the internal standard peptide and target peptide;

[0016] (4) A step of obtaining the peak area value of the target peptide obtained from the LC-MS analysis performed in the above step (3); and the peak area value of each internal standard peptide substituted with a stable isotope having a different amount;

[0017] (5) A step of obtaining an internal standard curve (Internal Standard Curve, Calibration Curve) formula from the area values ​​of internal standard peptides having different amounts obtained in the above step (4); and

[0018] (6) A method for identifying a target peptide based on mass spectrometry using a stable isotope-labeled internal standard peptide is provided, including a step of quantifying the area value of the target peptide obtained in step (4) by substituting the internal standard curve formula obtained in step (5).

[0019] The present invention relates to a mass spectrometry-based method for quantifying a target peptide using an internal standard peptide labeled with a stable isotope. The quantification method of the present invention, when quantifying a target peptide whose sequence is known, simultaneously generates an internal standard curve and analyzes the target peptide in a single analysis in a sample containing a mixture of several types of peptides (proteins), thereby enabling accurate quantification of each peptide.

[0020] Figure 1 is a schematic diagram of a method for quantitatively determining a peptide based on a stable isotope multi-labeled internal standard.

[0021] Figure 2 shows the LC results from LC-MS that simultaneously analyzed the target peptide (LASVADVAAK) and its stable isotope-multiply labeled internal standard peptide.

[0022] Figure 3 is an internal standard curve for the LASVADVAAK sequence established using the mass value of the internal standard peptide.

[0023] Figure 4 is a flow chart of a mass spectrometry-based method for quantifying a target peptide using a stable isotope-labeled internal standard peptide of the present invention.

[0024] The present invention comprises (1) a step of synthesizing multiple types of internal standard peptides having the same amino acid sequence as the target peptide, in which amino acids at both terminals or one terminal are substituted with stable isotopes, and in which the number of substituted amino acids is n or less when the number of amino acids of the target peptide to be quantified is n;

[0025] (2) A step of adding the internal standard peptide synthesized in step (1) to a sample containing the target peptide to be quantified, and mixing the amount of the internal standard peptide added in a proportional or inversely proportional amount according to the number of amino acids containing the substituted stable isotope;

[0026] (3) After the above step (2), a step of performing LC (Liquid Chromatography)-MS (Mass Spectrometry) or LC-MS / MS analysis on a sample containing the internal standard peptide and target peptide;

[0027] (4) A step of obtaining the peak area value of the target peptide obtained from the LC-MS analysis performed in the above step (3); and the peak area value of each internal standard peptide substituted with a stable isotope having a different amount;

[0028] (5) A step of obtaining an internal standard curve (Internal Standard Curve, Calibration Curve) formula from the area values ​​of internal standard peptides having different amounts obtained in the above step (4); and

[0029] (6) A method for quantifying a target peptide based on mass spectrometry using a stable isotope-labeled internal standard peptide, including a step of quantifying the area value of the target peptide obtained in step (4) by substituting the internal standard curve formula obtained in step (5) above (Fig. 4).

[0030] The above stable isotopes are 13C and 15 It is preferable that at least one of N be selected, but is not limited thereto.

[0031] In the above step (2), the amount of the internal standard peptide added is continuously and at a constant ratio, proportional or inversely proportional to the number of substituted stable isotopes, so that the mixing amount is increased by, for example, 10 times, 100 times, 1000 times, or 10000 times each time the number of substitutions increases, based on the internal standard peptide with 1 stable isotope substituted, or conversely, 10 -1 pear, 10 -2 pear, 10 -3 pear or 10 -4 The multiple can be increased, and in this way, the increasing multiple can be mixed in an amount that increases or decreases based on a certain standard. In addition, the substitution position is preferably based on one of the two ends, but any position may be used as a standard (Fig. 1).

[0032] The number of the target peptides is preferably at least one selected from 1 to 4,000, and the target peptide to be quantified is preferably a peptide composed of 6 to 80 amino acids, but is not limited thereto.

[0033] The target peptide to be quantified includes an internally cleaved peptide treated with a protease, and the protease is trypsin, chymotrypsin, pepsin, LysC or ArgC, but is not limited thereto.

[0034] In addition, the present invention comprises (1) a step of synthesizing multiple types of internal standard peptides having the same sequence as the target peptide, in which amino acids at both terminals or one terminal are substituted with stable isotopes, and in which the number of substituted amino acids is n or less when the number of amino acids of the target peptide to be determined is n;

[0035] (2) A step of adding the internal standard peptide synthesized in step (1) to a sample containing the target peptide to be determined, and mixing the internal standard peptide by adding an amount of the internal standard peptide that is proportional or inversely proportional to the number of amino acids containing the substituted stable isotope;

[0036] (3) After the above step (2), a step of performing LC (Liquid Chromatography)-MS (Mass Spectrometry) or LC-MS / MS analysis on a sample containing the internal standard peptide and target peptide;

[0037] (4) A step of obtaining the peak area value of the target peptide obtained from the LC-MS analysis performed in the above step (3); and the peak area value of each internal standard peptide substituted with a stable isotope having a different amount;

[0038] (5) A step of obtaining an internal standard curve (Internal Standard Curve, Calibration Curve) formula from the area values ​​of internal standard peptides having different amounts obtained in the above step (4); and

[0039] (6) A method for identifying a target peptide based on mass spectrometry using a stable isotope-labeled internal standard peptide, including a step of quantifying the area value of the target peptide obtained in step (4) by substituting it into the internal standard curve formula obtained in step (5). In the present invention, identification means identifying whether a target peptide is included in a specific mixture and the quantitative degree (quantitative value).

[0040]

[0041] Hereinafter, the present invention will be described in more detail using examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0042]

[0043] Example 1. Synthesis of an internal standard peptide consisting of the target peptide to be quantified and the same sequence, and multiply labeled with stable isotopes.

[0044] The target peptide (LASVADVAAK) shown in Table 1 was synthesized using a peptide synthesizer. Peptides of various lengths were synthesized using chemicals such as perfluoromoc (Fmoc) and tBoc. After removing the protecting group used in peptide synthesis, synthesis began in the N-direction, extending the peptide chain and coupling amino acids protected by other protecting groups. Subsequently, reactants, byproducts, protecting groups, and other impurities were removed, and after the final coupling reaction, the last amino acid not protected by Fmoc was oxidized to form a disulfide bond. The synthesized peptide was removed from the resin, and this step was repeated multiple times to synthesize peptides of the desired length.

[0045] Peptide Sequence Injection Amount Peak Area Value LASVADVAAK (Target Peptide) Unknown 699822 LASVADVAAK 1 pmol 82 LASVADVAAK 10 pmol 936 LASVADVAAK 100 pmol 1847 LASVADVAAK 1,000 pmol 8621 LASVADVAAK 10,000 pmol (1 nmol) 99727 LASVADVAAK 100,000 pmol (10 nmol) 869503 LASVADVAAK 1,000,000 pmol (100 nmol) 1763243 LASVADVAAK 10,000,000 pmol (1,000 nmol) 18209334

[0046]

[0047] Example 2. Design and synthesis of stable isotope multi-labeled internal standard peptides. A synthetic peptide labeled with a stable isotope complementary to the target peptide was used to synthesize the amino acid 12 C and 14 N each 13 C and 15 An internal standard peptide was synthesized using amino acids substituted with N (Stable Isotope-labeled Standard, SIS). In Table 1 above, stable isotopes are underlined and bolded, and the mixed amount was mixed so that the amount increased by 10 times for each increase in the number of stable isotopes substituted.

[0048] Meanwhile, as disclosed in Table 2, the mass values ​​of amino acids substituted with stable isotopes and those that were not substituted were calculated. In this way, if the sequence of a peptide or protein is known, the mass value of the peptide according to the degree of stable isotope substitution can be theoretically calculated, and the peptide sequence can be mapped (specified) from the experimental mass value obtained from the mass spectrometry result in comparison to such calculated value.

[0049] Differences in mass values ​​due to stable isotope substitutionAmino acid types 13 C and 15 Difference in the mass value of the monoisotopic substituted with N 13C-substituted Monoisotopic mass value difference 15N이 치환된Monoisotopic질량 값 차이Alanine (Ala, A)4.007099 Da3.010065 Da0.997035 DaCysteine ​​(Cys, C)4.007099 Da3.010065 Da0.997035 DaAspartic Acid (Asp, D)5.010454 Da4.013419 Da0.997035 DaGlutamic Acid (Glu, E)6.013809 Da5.016774 Da0.997035 DaPhenylalanine (Phe, F)10.027228 Da9.030194 Da0.997035 DaGlycine (Gly, G)3.003745 Da2.00671 Da0.997035 DaHistidine (His, H)9.011234 Da6.020129 Da2.991105 DaIsoleucine (Ile, I)7.017164 Da6.020129 Da0.997035 DaLysine (Lys, K)8.014199 Da6.020129 Da1.99407 DaLeucine (Leu, L)7.017164 Da6.020129 Da0.997035 DaMethionine (Met, M)6.013809 Da5.016774 Da0.997035 DaAsparagine (Asn, N)6.007489 Da4.013419 Da1.99407 DaProline (Pro, P)6.013809 Da5.016774 Da0.997035 DaGlutamine (Gln, Q)7.010844 Da5.016774 Da1.99407 DaArginine (Arg, R)10.008269 Da6.020129 Da3.98814 DaSerine (Ser, S)4.007099 Da3.010065 Da0.997035 DaThreonine (Thr, T)5.010454 Da4.013419 Da0.997035 DaValine (Val, V)6.013809 Da5.016774 Da0.997035 DaTryptophan (Trp, W)13.030973 Da11.036903 Da1.99407 DaTyrosine (Tyr, Y)10.027228 Da9.030194 Da0.997035 Da.

[0050] In this Example 1, for quantification of the LASVADVAAK sequence, 13 C and 15 After sequentially synthesizing different amounts (concentrations) of internal standard peptides according to the number of amino acids substituted with N (stable isotope-substituted amino acids are underlined), a mixture of these was prepared. The difference in monoisotopic mass values ​​between stable isotope-labeled and unlabeled amino acids was calculated. The mass values ​​of the peptide mass (MS1, precursor ion) and the mass values ​​of the fragmented peptide (MS2, product ion) were calculated.

[0051] A stable isotope-labeled internal standard was added to a sample containing the target peptide to be analyzed. The target peptide and the stable isotope-labeled internal standard with different mass values ​​were simultaneously analyzed by LC-MS.

[0052] As a result, as disclosed in Fig. 2, the target peptide (one) and eight synthetic peptides labeled with stable isotopes in different forms had identical physicochemical properties and were detected simultaneously at the same retention time (RT).

[0053] The analytical values ​​of the target peptide and the analytical values ​​of the internal standard were calculated as peak area values, and then the analytical values ​​of the internal standard having different amounts were used to obtain the internal standard curve (Internal Standard Curve, Calibration Curve) equation (1) (Fig. 3).

[0054] The analysis value (peak area) of the target peptide was substituted into the internal standard curve equation to finally calculate the amount (concentration) of the target peptide.

[0055] Internal standard curve equation (1)

[0056] y = 1.8094 x + 106140

[0057] In the above equation (1), y is the peak area, and x is the amount (concentration) of the sample (peptide) to be analyzed.

[0058] Therefore, by substituting the peak area (699822) obtained in the example of the present invention, it was confirmed that the amount of peptide was 328,109.87 pmol.

Claims

1. (1) When the number of amino acids of a target peptide to be quantified is n, a step of synthesizing multiple types of internal standard peptides having the same amino acid sequence as the target peptide, in which amino acids at both terminals or one terminal are substituted with stable isotopes, and in which the number of substituted amino acids is n or less; (2) A step of adding the internal standard peptide synthesized in step (1) to a sample containing the target peptide to be quantified, wherein the amount of the internal standard peptide added is proportional or inversely proportional to the number of amino acids containing the substituted stable isotope, and mixing the same; (3) After the step (2), a step of performing LC (Liquid Chromatography)-MS (Mass Spectrometry) or LC-MS / MS analysis on a sample containing the internal standard peptide and target peptide; (4) a step of obtaining the peak area value of the target peptide obtained from the LC-MS analysis performed in the above step (3); and a step of obtaining the peak area value of each internal standard peptide substituted with a stable isotope having a different amount; (5) A step of obtaining an internal standard curve (calibration curve) formula from the area values ​​of internal standard peptides having different amounts obtained in the above step (4); and (6) A method for quantifying a target peptide based on mass spectrometry using a stable isotope-labeled internal standard peptide, comprising: a step of quantifying by substituting the area value of the target peptide obtained in step (4) into the internal standard curve formula obtained in step (5); 2. In paragraph 1, the stable isotope is 13 C and 15 A mass spectrometry-based method for quantifying a target peptide using a stable isotope-labeled internal standard peptide characterized by at least one selected from N.

3. A mass spectrometry-based method for quantifying target peptides using a stable isotope-labeled internal standard peptide, characterized in that the number of target peptides in paragraph 1 is at least one selected from 1 to 4,000.

4. A mass spectrometry-based method for quantifying a target peptide using a stable isotope-labeled internal standard peptide, characterized in that the target peptide to be quantified in the first paragraph is a peptide consisting of 6 to 80 amino acids.

5. A mass spectrometry-based method for quantifying a target peptide using a stable isotope-labeled internal standard peptide, characterized in that the target peptide to be quantified in the first paragraph is a peptide that has been internally cleaved by treating with a protease.

6. A mass spectrometry-based method for quantifying a target peptide using a stable isotope-labeled internal standard peptide, characterized in that in paragraph 5, the protease is trypsin, chymotrypsin, pepsin, LysC or ArgC. 7.(1) A step of synthesizing multiple types of internal standard peptides having the same sequence as the target peptide, in which amino acids at both terminals or one terminal are substituted with stable isotopes, and the number of substituted amino acids is n or less, when the number of amino acids of the target peptide to be determined is n; (2) A step of adding the internal standard peptide synthesized in step (1) to a sample containing the target peptide to be determined, wherein the amount of the internal standard peptide added is proportional or inversely proportional to the number of amino acids containing the substituted stable isotope, and mixing the internal standard peptide; (3) After the step (2), a step of performing LC (Liquid Chromatography)-MS (Mass Spectrometry) or LC-MS / MS analysis on a sample containing the internal standard peptide and target peptide; (4) a step of obtaining the peak area value of the target peptide obtained from the LC-MS analysis performed in the above step (3); and a step of obtaining the peak area value of each internal standard peptide substituted with a stable isotope having a different amount; (5) A step of obtaining an internal standard curve (calibration curve) formula from the area values ​​of internal standard peptides having different amounts obtained in the above step (4); and (6) A method for identifying a target peptide based on mass spectrometry using a stable isotope-labeled internal standard peptide, comprising: a step of quantifying the area value of the target peptide obtained in step (4) by substituting it into the internal standard curve formula obtained in step (5);

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