CN114577916B - Analysis method of stable isotope labeling metabolic flow based on chip nanoliter electrospray mass spectrum - Google Patents
Analysis method of stable isotope labeling metabolic flow based on chip nanoliter electrospray mass spectrum Download PDFInfo
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Abstract
The invention discloses an analysis method of stable isotope labeling metabolic flow based on a chip nanoliter electrospray mass spectrum. The metabolites in the cells are directly extracted by adopting methanol/water, transferred to a sample injection disc, added with a spraying solvent, and directly injected into a mass spectrometer for analysis through a chip-based multichannel nano-liter electrospray ion source. And obtaining the metabolite information marked by the stable isotope by adopting a full scanning and parallel reaction monitoring mode and combining a self-programmed secondary mass spectrum matching operation program. And quantifying the stable isotope labeled metabolites in the sample by adopting a parallel reaction monitoring mode. The analysis method can carry out quantitative analysis on the stable isotope labeled metabolites in a small amount of cells, and the analysis time is greatly shortened by direct sample injection. The method has the advantages of simple pretreatment, economy, high efficiency, high sensitivity and high flux.
Description
Technical Field
The invention relates to the fields of analytical chemistry and stable isotope labeling and tracing, in particular to an analysis method for quantitatively stabilizing isotope labeling metabolic flow by adopting a parallel reaction monitoring mode based on a chip nanoliter electrospray mass spectrum, a labeled metabolite is obtained according to matching of a primary mass spectrum and a secondary mass spectrum.
Background
Metabolic reprogramming is an important feature of a variety of diseases. Traditional metabonomics analysis can only reflect static metabolite concentration information, and lacks dynamic process information of biochemical reaction. Based on a stable isotope tracing method and assisted dynamic process information analysis, the change of metabolites in organisms and the functional verification of metabolic pathways can be known, and the method can be used for exploring the occurrence mechanism and treatment scheme of diseases.
The existing quantitative method for the stable isotope labeling metabolic flux comprises gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry and capillary electrophoresis-mass spectrometry. These methods require millions of cells and are not suitable for rare samples. One culture requires about 10mL of a substrate labeled with a stable isotope, and the substrate labeled with a stable isotope is expensive and uneconomical. In addition, the analysis time is long. In conclusion, it is necessary to develop a high-throughput stable isotope-labeled metabolic flux assay for small samples.
The research adopts a chip-based multichannel nanoliter electrospray ion source to directly inject a sample into a mass spectrometer for analysis, and only about 10000 cells and 100 mu L of culture medium are needed for analysis at least once. The method can be used for analyzing rare samples, reduces the cost of culture medium, and is more economical. In addition, the pretreatment method is simple and convenient, the analysis time is short, and the method is favorable for the analysis of large-scale samples. There have been no reports to date of the application of the method of the present invention to stable isotope-labeled metabolic flux analysis.
Disclosure of Invention
In order to realize stable isotope labeled metabolic flow, the invention establishes an analysis method based on chip nanoliter electrospray mass spectrometry and adopting a parallel reaction monitoring mode to quantify the stable isotope labeled metabolic flow. The method has the advantages of simple pretreatment, economy and colleges, small required sample size, short analysis time, comprehensive information acquisition and the like.
The specific technical method adopted by the invention is as follows:
(1) The cells are cultured in two media, one stable isotope-labeled medium containing stable isotope-labeled metabolites, e.g. 13 C 6 -a glucose, 13 c-1,2-glucose, 13 C 3 -pyruvic acid, 13 C 5 -a source of glutamine, 15 N 2 -a source of glutamine, 13 C 5 - 15 N 2 -a source of glutamine, 2 H 3 -serine, etc.; the other is an unstable isotope labeled medium which contains metabolites that are not labeled with stable isotopes and at a concentration consistent with the stable isotope labeled metabolites in the stable isotope labeled medium. Cells are cultured in 6-well, 12-well, 24-well, 96-well or 10cm culture plates, and the cell density of one well reaches 50% -90%. The medium was discarded and washed twice with PBS. Counting the cells, adding 50-100 μ L of 80% methanol/water extracting agent containing 5-20 μ M methionine sulfone and camphorsulfonic acid sodium salt as internal standard per 10000-20000 cells. Covering with aluminum foil paper and sealing film, and standing at 3-5 deg.C for 30-40min.
(2) Transfer 20-40 μ L of extract to 96-well template and add equal volume of spray solvent. In the positive ion detection mode, the spray solvent is 60-80% methanol/water (v/v) containing 0.3-0.5% formic acid; in the negative ion detection mode, the spray solvent is 60-80% methanol/water (v/v) containing 10-30mM ammonium acetate and 0.03-0.05% formic acid. The ions are directly injected into the mass spectrometer through a chip-based multichannel nanoliter electrospray ion source.
(3) And performing mass spectrometry on the cell extracting solution cultured in the stable isotope labeling culture medium and the cell extracting solution cultured in the non-stable isotope labeling culture medium by adopting a full-scanning monitoring mode to obtain first-level intensity information. And filtering metabolites with the signal-to-noise ratio of less than 3, which appear in less than 60-80% of repeated samples for multiple times and exist in blank by primary mass spectrum filtration to obtain a characteristic ion table stably existing in the unlabeled and labeled cell extracting solution. Wherein the blank sample refers to a blank solvent without containing the cell sample, namely a mixed solution of an extracting agent and a spraying solvent. According to the precise mass number (abbreviated as M) of the characteristic ions stably existing in the unmarked part, the precise mass number (abbreviated as M + n, n refers to the number of atoms marked by the stable isotope) of the corresponding isotope on the mark is calculated and matched with the characteristic ions stably existing in the marked cell extracting solution. If there is a match, the metabolite is considered to be a potential labeled metabolite. If not, neglect.
(4) And (3) carrying out parallel reaction monitoring mode mass spectrometry on the cell extracting solution cultured in the stable isotope labeling culture medium and the cell extracting solution cultured in the non-stable isotope labeling culture medium aiming at the potential labeled metabolites to obtain secondary mass spectrum information of the cell extracting solution. In a stable isotope labeled sample, secondary mass spectrograms of accurate mass numbers M and M + n are respectively extracted, all secondary ions (abbreviated as M → Mp) are generated according to M, the mass number of the secondary ions labeled by the stable isotope is calculated, and the secondary mass spectrograms are matched with the secondary mass spectrograms of M + n (abbreviated as M + n → Mp + M, M refers to the number of atoms labeled by the stable isotope, and M is less than or equal to n). The matching rules are equal at a mass deviation of 5-20 ppm. If not, it is ignored. And if the intensities are matched, calculating the ratio of the Mp + m intensity to the Mp intensity. Meanwhile, M → Mp and M + n → Mp + M are extracted from the sample without stable isotope labeling, the ratio of the intensity of Mp + M to the intensity of Mp is calculated, and if the value obtained by multiplying the ratio of the intensity of Mp + M to the intensity of Mp in the sample without stable isotope labeling by 1.0-1.2 is smaller than the ratio of the intensity of Mp + M to the intensity of Mp in the sample with stable isotope labeling, M and M + n are considered as a pair of non-labeled and labeled metabolites, and respectively contain secondary fragments Mp and Mp + M. According to the above rules, stable isotope labeled metabolites were found, and their primary and secondary mass spectral information was used for qualitative and quantitative analysis.
(5) And obtaining the ion pairs of the labeled metabolites through the primary and secondary mass spectrum information of the labeled metabolites, and carrying out quantitative analysis by adopting a parallel reaction monitoring mode. The conditions for the parallel reaction monitoring mode were: the maximum ion collection time is 100-200 ms, each 0.1-0.3 s is a time period, and no more than 50 ions are contained in one time period, so that at least 5 points are collected for each ion. And simultaneously, collecting the intensity of the internal standard in a whole section. For each ion, the intensity was divided by the internal standard intensity at the same time, and the average of 5 points was taken as a semi-quantitative result.
The method only needs 10000 cells and 10 mu L of culture medium at least, is more economical and can be used for analyzing rare samples; the extraction solution is directly injected into the mass spectrum by adopting a chip nanoliter electrospray ion source, and compared with the traditional method for combining the chromatographic mass spectrum, the sensitivity is improved, and the analysis time is shortened; and acquiring primary and secondary mass spectrum information by adopting a full-scanning and parallel reaction monitoring mode, and after accurate mass number and intensity screening is carried out, finding out the labeled metabolite to acquire comprehensive primary and secondary mass spectrum information for labeling the metabolite.
Drawings
FIG. 1 illustrates a two-stage mass matching process, exemplified by 116.0705 and 121.0872.
FIG. 2 metabolic changes of isocitrate dehydrogenase mutant U251 human glioma cells under the action of triptolide.
Table 1 positive ion mode, potential labeled ions.
Table 2 negative ion mode, potential labeled ions.
Table 3 labeled ions and their secondary mass spectral information in positive ion mode.
Table 4 information of labeled ions and their secondary mass spectra in negative ion mode.
Detailed Description
Embodiments of the invention are described in detail below with reference to the attached table drawings: the embodiments are implemented on the premise of the technical scheme of the invention, and detailed implementation modes and specific operation processes are given, but the protection scope of the invention is not limited by the following embodiments.
Example one
Based on chip nanoliter electrospray mass spectrometry, full-scan and parallel reaction monitoring mode analysis, the carbon source metabolic flow of glutamine is analyzed.
(1) Cell culture and pretreatment: u251 cells were cultured in 96-well culture plates to a density of 90%, i.e., about 10000 cells per well. The culture medium is Du's medium (DMEM) containing 4mM glutamine, and the culture medium contains stable isotope labeled glutamine (4 mM) 13 C 5 -glutamine) DMEM. After 1 day of incubation, the medium was discarded and washed twice with PBS. 50 μ L of 80% methanol/water extract containing 10 μ M methionine sulfone and camphorsulfonic acid sodium salt as internal standard was added. Covering with aluminum foil paper and sealing film, and standing at 4 deg.C for 30min. Each sample was replicated 4 times.
(2) Transfer 20. Mu.L of each extract to 2 96-well plates, add 20. Mu.L of 80% methanol/water (v/v) containing 0.5% formic acid (v/v) to one plate for positive ion mode detection; another plate was loaded with 20. Mu.L of 80% methanol/water (v/v) containing 20mM ammonium acetate and 0.04% formic acid (v/v) for negative ion mode detection. The ions are directly injected into the mass spectrometer through a chip-based multichannel nanoliter electrospray ion source.
(3) Full scan mode mass spectrometry conditions: ESI source, positive ion mode and negative ion mode.
(4) And filtering according to the first-class mass spectrum information, and filtering metabolites which are present in a blank and have the signal-to-noise ratio of less than 3 and appear in the quadruplicate samples for less than three times to obtain a characteristic ion table stably existing in the unlabeled and labeled cell extracting solution. According to the precise mass number (abbreviated as M) of the characteristic ions stably existing in the unmarked part, the precise mass number (abbreviated as M + n, n refers to the number of atoms marked by the stable isotope) of the corresponding isotope on the mark is calculated and matched with the characteristic ions stably existing in the marked cell extracting solution. If they match, they are considered as potential labeled metabolites, and the potential mass numbers are shown in attached tables 1 and 2. If not, neglecting
(5) Parallel reaction monitoring mode mass spectrometry conditions: ESI source, positive ion mode and negative ion mode, collision energy is 15eV,30eV,45eV. And (4) aiming at the potential labeled metabolites in the step (4), carrying out parallel reaction monitoring mode analysis on the cell extract cultured in the stable isotope labeled culture medium and the cell extract cultured in the non-stable isotope labeled culture medium to obtain the second-order mass spectrum information of all ions.
(6) From the secondary mass spectral information, the labeled metabolites were confirmed: in a stable isotope labeled sample, extracting secondary mass spectrograms of accurate mass numbers M and M + n respectively, generating all secondary ions (abbreviated as M → Mp) according to M, calculating the mass numbers of the secondary ions labeled by the stable isotope, namely M, M +1 x 1.003355, M +2 x 1.003355, M +3 x 1.003355, M +4 x 1.003355, M +5 x 1.003355, and matching with the secondary mass spectrograms of M + n (abbreviated as M + n → Mp + M, n and M refer to the number of atoms labeled by the stable isotope, wherein M is not more than n). The matching rule is at 10Mass deviation in ppm is equal. If not, neglect. And if the intensities are matched, calculating the ratio of the Mp + m intensity to the Mp intensity. Meanwhile, M → Mp and M + n → Mp + M are extracted from the sample not containing stable isotope labeling, the ratio of the intensity of Mp + M to the intensity of Mp is calculated, and if it is smaller than the ratio of the intensity of Mp + M to the intensity of Mp in the sample labeled with stable isotope, M and M + n are considered as a pair of unlabeled and labeled metabolites, containing secondary fragments Mp and Mp + M, respectively. Taking the potentially labeled ions with M and M + n of 116.0705 and 121.0872 as an example, the specific process is shown in FIG. 1. Firstly, firstly 13 C 5 Glutamine-labeled samples, accurate mass number plus 0,1 x 1.03355,2 x 1.03355,3 x 1.03355,4 x 1.03355,5 x 1.03355 for each secondary fragment of 116.0705, and secondary fragments of 121.0872 were searched for the presence of ions with accurate mass within 5 ppm. Four pairs of ions were found with an exact mass number difference of N1.003355 with N =0,1,2,3,4,5. The intensity ratio of the secondary fragments is calculated and is recorded as ratio _13. Second, in the unlabeled samples, the secondary patch intensities of the corresponding 116.0705 and 121.0872 are extracted, and the ratio _12 is calculated. A secondary fragment is considered to be its secondary fragment when its ratio _13 is greater than 1.1 times ratio _12 and the absolute intensity of the secondary fragment in the marked sample is greater than it is in the unmarked sample. In FIG. 1, the limit of ratio _12, where ratio _13 is greater than 1.1 times, excludes 74.0598 and 75.0632; the absolute intensity of the secondary fragment in the labeled sample was greater than its limit in the unlabeled sample, excluding 56.0495 and 57.0530. For the potential labeled metabolites of 1160705 and 121.0872, the corresponding 70.0650 and 74.0784 fragments were found. According to the above rules, stable isotope labeled metabolites were found, and their primary and secondary mass spectral information was used for qualitative and quantitative purposes.
(7) Characterization of the labeled metabolites: the labeled metabolites were characterized according to the HMDB database, in combination with the existing secondary information. Taking 1116.0705 in step (6) as an example, it contains 70.0650 characteristic fragment, and it is presumed to be proline from the database.
Finally, in positive ion mode 55 labelled metabolites were detected, 13 of which were characterised. In negative ion mode, 35 labeled metabolites were detected, 16 of which were characterized. The information and qualitative information of the primary and secondary mass spectra of the labeled metabolites is shown in attached table 3 and attached table 4.
Example two
Isocitrate dehydrogenase I mutated human glioma cells (U251 IDH 1) RH ) Glutamine metabolism pathway change under action of triptolide
(1) Cell culture and pretreatment: u251 cells were cultured in 96-well culture plates to a density of 70%, i.e., approximately 10000 cells per well. The culture medium contains stable isotope labeled glutamine (4 mM) 13 C 5 -glutamine) DMEM. Adding 0nM and 120nM triptolide, and culturing for 12h. The medium was discarded and washed twice with PBS. 50 μ L of 80% methanol/water extractant containing 5-20 μ M methionine sulfone and camphorsulfonic acid sodium salt as internal standard was added. Covering with aluminum foil paper and sealing film, and standing at 4 deg.C for 30min.
(2) Transferring 20 μ L of the extract to 2 96-well sample plates, one of which is added 20 μ L of 80% methanol/water containing 0.5% formic acid for positive ion mode detection; the other plate was charged with 20. Mu.L of 80% methanol/water containing 20mM ammonium acetate and 0.04% formic acid for negative ion mode detection. The ions are directly injected into the mass spectrometer through a chip-based multichannel nanoliter electrospray ion source.
(3) And carrying out parallel reaction monitoring mode analysis on the labeled metabolites to obtain secondary information for quantification. Mass spectrum conditions: ESI source, positive ion mode and negative ion mode, collision energy of 15ev,30ev,45ev, maximum ion collection time of 200ms, a time period of about 12 ions per 0.2s, contained in one time period to ensure that at least 5 points were collected per ion.
(4) For each ion, the intensity was divided by the internal standard intensity at the same time, and the average of 5 points was taken as a semi-quantitative result. Using the Mann-Whitney rank sum test, triptolide treated and untreated U251 IDH1 were found RH Metabolites with significant differences among cells are shown in fig. 2, and in fig. 2, blue is a metabolite from which glutamine is significantly reduced after treatment with triptolide. Black is noThere are significant differences. The triptolide can influence U251 IDH1 by inhibiting glutathione synthesis and tricarboxylic acid cycle RH Metabolism of the cell.
TABLE 1 potential labeled ions in Positive ion mode
| No. | M | M+1 | M+2 | M+3 | M+4 | M+5 | No. | M | M+1 | M+2 | M+3 | M+4 | M+5 |
| 1 | 103.0388 | 107.0522 | 108.0555 | 63 | 248.9987 | 254.0154 | |||||||
| 2 | 103.9735 | 108.99 | 64 | 249.9826 | 252.9927 | 254.9994 | |||||||
| 3 | 105.0545 | 110.0712 | 65 | 250.9667 | 255.9833 | ||||||||
| 4 | 107.0701 | 108.0735 | 112.0868 | 66 | 250.9951 | 256.0127 | |||||||
| 5 | 114.0661 | 116.0729 | 119.083 | 67 | 251.9797 | 256.9964 | |||||||
| 6 | 114.0733 | 119.0895 | 68 | 259.0276 | 264.0442 | ||||||||
| 7 | 116.0525 | 120.0654 | 121.0688 | 69 | 264.9728 | 269.9893 | |||||||
| 8 | 116.0705 | 117.0738 | 118.0772 | 119.0806 | 120.0839 | 121.0873 | 70 | 265.9567 | 270.9733 | ||||
| 9 | 119.0806 | 120.0839 | 121.0873 | 124.0968 | 71 | 267.9538 | 272.9702 | ||||||
| 10 | 124.9999 | 130.0168 | 72 | 271.9646 | 274.9745 | 276.9817 | |||||||
| 11 | 130.0498 | 131.0532 | 133.0601 | 134.0632 | 135.0666 | 73 | 272.9487 | 277.9654 | |||||
| 12 | 131.0563 | 136.0733 | 74 | 273.9618 | 278.9785 | ||||||||
| 13 | 132.0768 | 133.0801 | 137.0936 | 75 | 274.9456 | 279.9625 | |||||||
| 14 | 133.097 | 138.1139 | 76 | 275.0015 | 280.018 | ||||||||
| 15 | 139.0155 | 144.0323 | 77 | 281.9935 | 287.0112 | ||||||||
| 16 | 145.0471 | 149.0602 | 150.0645 | 78 | 282.9776 | 287.9937 | |||||||
| 17 | 147.0764 | 148.0796 | 151.0897 | 152.0931 | 79 | 285.9672 | 290.9849 | ||||||
| 18 | 148.0604 | 149.0637 | 150.0671 | 151.0703 | 152.0737 | 153.0771 | 80 | 287.9385 | 292.9552 | ||||
| 19 | 149.0573 | 154.0741 | 81 | 288.9223 | 293.9391 | ||||||||
| 20 | 149.0808 | 154.0973 | 82 | 289.017 | 294.0335 | ||||||||
| 21 | 150.0646 | 155.0815 | 83 | 289.9357 | 294.9526 | ||||||||
| 22 | 151.0479 | 154.0585 | 156.0644 | 84 | 290.0009 | 295.018 | |||||||
| 23 | 152.0318 | 153.035 | 155.0422 | 157.0485 | 85 | 290.985 | 296.0017 | ||||||
| 24 | 153.0158 | 157.0299 | 158.0333 | 86 | 305.9751 | 310.9915 | |||||||
| 25 | 156.0767 | 161.0935 | 87 | 306.9579 | 311.974 | ||||||||
| 26 | 161.0692 | 166.0862 | 88 | 307.0829 | 312.0999 | ||||||||
| 27 | 162.076 | 167.0929 | 89 | 308.0907 | 311.1007 | 312.104 | 313.1074 | ||||||
| 28 | 166.071 | 171.0877 | 90 | 309.0945 | 312.104 | 313.1074 | 314.1108 | ||||||
| 29 | 169.0583 | 170.0616 | 173.0717 | 174.075 | 91 | 310.0863 | 315.1031 | ||||||
| 30 | 170.0423 | 171.0456 | 172.049 | 173.0524 | 174.0558 | 175.0591 | 92 | 310.0956 | 315.1123 | ||||
| 31 | 170.0924 | 175.1091 | 93 | 310.9989 | 316.0155 | ||||||||
| 32 | 171.0263 | 172.0296 | 173.0331 | 174.0364 | 175.0398 | 176.0431 | 94 | 311.9828 | 316.9995 | ||||
| 33 | 171.0304 | 176.0472 | 95 | 312.9668 | 317.9838 | ||||||||
| 34 | 173.0305 | 178.0474 | 96 | 327.9567 | 332.9732 | ||||||||
| 35 | 173.0421 | 178.0586 | 97 | 329.9232 | 334.9397 | ||||||||
| 36 | 173.1171 | 178.1338 | 98 | 330.0726 | 331.076 | 332.079 | 333.0826 | 334.0858 | 335.0892 | ||||
| 37 | 175.1189 | 176.1222 | 180.1357 | 99 | 330.9069 | 335.9238 | |||||||
| 38 | 176.0658 | 181.0833 | 100 | 331.0763 | 334.0858 | 335.0892 | 336.0929 | ||||||
| 39 | 176.103 | 181.1196 | 101 | 332.0682 | 335.0782 | 337.0849 | |||||||
| 40 | 185.0211 | 190.0374 | 102 | 333.9648 | 338.9822 | ||||||||
| 41 | 185.0322 | 190.0489 | 103 | 334.9486 | 339.965 | ||||||||
| 42 | 186.0162 | 189.0262 | 191.0329 | 104 | 336.0856 | 337.0888 | 339.0958 | 341.102 | |||||
| 43 | 186.9677 | 191.9854 | 105 | 338.1014 | 343.118 | ||||||||
| 44 | 187.0002 | 188.0035 | 190.0102 | 192.0169 | 106 | 339.0772 | 344.0933 | ||||||
| 45 | 187.0304 | 189.0372 | 192.047 | 107 | 345.9705 | 350.9868 | |||||||
| 46 | 189.0158 | 194.0318 | 108 | 346.0469 | 349.0567 | 351.0634 | |||||||
| 47 | 191.0401 | 196.0569 | 109 | 349.9386 | 354.9553 | ||||||||
| 48 | 192.0241 | 193.0274 | 195.0342 | 196.0375 | 197.0409 | 110 | 352.0544 | 355.0646 | 356.0675 | 357.0711 | |||
| 49 | 193.0083 | 196.0182 | 198.0249 | 111 | 354.0962 | 357.106 | 358.1096 | 359.1127 | |||||
| 50 | 196.0571 | 201.0732 | 112 | 355.0996 | 357.106 | 358.1096 | 359.1127 | 360.116 | |||||
| 51 | 200.0404 | 205.0567 | 113 | 356.0915 | 361.1082 | ||||||||
| 52 | 205.0335 | 210.05 | 114 | 358.0669 | 363.0843 | ||||||||
| 53 | 207.0141 | 212.0308 | 115 | 360.0467 | 365.0636 | ||||||||
| 54 | 207.9981 | 211.0082 | 213.0148 | 116 | 368.0288 | 373.0452 | |||||||
| 55 | 208.9821 | 211.9921 | 213.9988 | 117 | 368.1117 | 371.1217 | 372.1254 | 373.1285 | |||||
| 56 | 212.0891 | 217.1066 | 118 | 369.1148 | 371.1217 | 372.1254 | 373.1285 | 374.1319 | |||||
| 57 | 215.0512 | 220.0675 | 119 | 374.0363 | 377.0464 | 379.0531 | |||||||
| 58 | 216.0144 | 221.0303 | 120 | 376.0779 | 377.0814 | 379.088 | 381.0945 | ||||||
| 59 | 217.0192 | 219.0263 | 220.0298 | 222.035 | 121 | 390.0103 | 395.0272 | ||||||
| 60 | 224.1257 | 229.1415 | 122 | 391.9229 | 396.9404 | ||||||||
| 61 | 229.9801 | 234.9967 | 123 | 398.0599 | 403.0766 | ||||||||
| 62 | 230.9641 | 235.9808 | 124 | 402.1191 | 407.1353 |
TABLE 1. Potential labeled ions in positive ion mode
| No. | M | M+1 | M+2 | M+3 | M+4 | M+5 | No. | M | M+1 | M+2 | M+3 | M+4 | M+5 |
| 125 | 420.04 | 425.06 | 189 | 275.99 | 280 | ||||||||
| 126 | 432 | 437.01 | 190 | 297.97 | 301.98 | ||||||||
| 127 | 450.03 | 455.05 | 191 | 311.09 | 315.1 | ||||||||
| 128 | 70.065 | 74.078 | 192 | 322.11 | 326.12 | ||||||||
| 129 | 74.024 | 78.037 | 193 | 353.06 | 355.06 | 356.07 | 357.07 | ||||||
| 130 | 84.044 | 86.051 | 88.058 | 194 | 375.04 | 377.05 | 379.05 | ||||||
| 131 | 89.107 | 93.121 | 195 | 377.08 | 379.09 | 381.09 | |||||||
| 132 | 95.031 | 99.044 | 196 | 88.039 | 91.049 | ||||||||
| 133 | 101.06 | 102.06 | 105.07 | 197 | 90.055 | 91.058 | 93.065 | ||||||
| 134 | 101.07 | 105.08 | 198 | 102.06 | 105.07 | ||||||||
| 135 | 102.05 | 103.06 | 104.06 | 106.07 | 199 | 103.06 | 104.06 | 106.07 | |||||
| 136 | 108.07 | 112.09 | 200 | 107.05 | 110.06 | ||||||||
| 137 | 110.06 | 114.07 | 201 | 120 | 123.01 | ||||||||
| 138 | 111.01 | 115.02 | 202 | 121.09 | 124.1 | ||||||||
| 139 | 112.98 | 117 | 203 | 131.07 | 134.08 | ||||||||
| 140 | 116.03 | 120.05 | 204 | 135.05 | 136.05 | 137.05 | 138.06 | ||||||
| 141 | 117.07 | 118.08 | 119.08 | 120.08 | 121.09 | 205 | 137.03 | 139.04 | 140.04 | ||||
| 142 | 121.04 | 123.04 | 125.05 | 206 | 138.05 | 141.06 | |||||||
| 143 | 131.05 | 133.06 | 134.06 | 135.07 | 207 | 147.17 | 148.17 | 150.18 | |||||
| 144 | 133.06 | 137.07 | 208 | 150.07 | 153.08 | ||||||||
| 145 | 133.08 | 137.09 | 209 | 157.03 | 158.03 | 159.04 | 160.04 | ||||||
| 146 | 134.04 | 135.05 | 136.05 | 137.05 | 138.06 | 210 | 173.09 | 176.1 | |||||
| 147 | 137.06 | 138.06 | 141.07 | 211 | 184.06 | 185.06 | 186.06 | 187.07 | |||||
| 148 | 139.04 | 140.04 | 143.05 | 212 | 184.56 | 185.56 | 186.57 | 187.57 | |||||
| 149 | 141.02 | 145.03 | 213 | 185.06 | 188.07 | ||||||||
| 150 | 146.17 | 147.17 | 148.17 | 150.18 | 214 | 189.04 | 192.05 | ||||||
| 151 | 148.08 | 151.09 | 152.09 | 215 | 204.23 | 205.23 | 207.24 | ||||||
| 152 | 149.06 | 150.07 | 151.07 | 152.07 | 153.08 | 216 | 214.01 | 217.02 | |||||
| 153 | 156.03 | 157.03 | 158.03 | 159.04 | 160.04 | 217 | 214.99 | 218 | |||||
| 154 | 156.95 | 160.97 | 218 | 311.1 | 312.1 | 313.11 | 314.11 | ||||||
| 155 | 157.01 | 159.02 | 161.02 | 219 | 81.031 | 83.037 | |||||||
| 156 | 157.01 | 161.03 | 220 | 83.022 | 85.028 | ||||||||
| 157 | 159.1 | 163.12 | 221 | 91.058 | 93.065 | ||||||||
| 158 | 160.92 | 164.93 | 222 | 105 | 107.01 | ||||||||
| 159 | 170.06 | 173.07 | 174.08 | 223 | 110.01 | 112.02 | |||||||
| 160 | 171.04 | 175.06 | 224 | 111.02 | 113.02 | ||||||||
| 161 | 171.05 | 172.05 | 173.05 | 174.06 | 175.06 | 225 | 111.05 | 113.06 | |||||
| 162 | 172 | 176.01 | 226 | 119.02 | 120.02 | 121.03 | |||||||
| 163 | 172.03 | 173.03 | 174.04 | 175.04 | 176.04 | 227 | 123.01 | 125.02 | |||||
| 164 | 178.01 | 179.01 | 180.02 | 182.02 | 228 | 123.04 | 125.05 | ||||||
| 165 | 178.99 | 183.01 | 229 | 133.03 | 135.04 | ||||||||
| 166 | 186.04 | 190.05 | 230 | 136.05 | 137.05 | 138.06 | |||||||
| 167 | 187.02 | 189.03 | 191.03 | 231 | 139.06 | 141.06 | |||||||
| 168 | 187.04 | 191.05 | 232 | 142.03 | 144.04 | ||||||||
| 169 | 188 | 190.01 | 192.02 | 233 | 147.05 | 149.06 | |||||||
| 170 | 192.04 | 196.06 | 234 | 151.05 | 153.05 | ||||||||
| 171 | 193.03 | 195.03 | 196.04 | 197.04 | 235 | 151.07 | 152.07 | 153.08 | |||||
| 172 | 193.98 | 198 | 236 | 167.06 | 169.06 | ||||||||
| 173 | 194.01 | 196.02 | 198.02 | 237 | 174.04 | 175.04 | 176.04 | ||||||
| 174 | 198.04 | 202.05 | 238 | 185.06 | 186.06 | 187.07 | |||||||
| 175 | 199.99 | 204 | 239 | 215.02 | 217.02 | ||||||||
| 176 | 201.11 | 205.13 | 240 | 239.15 | 241.16 | ||||||||
| 177 | 203.22 | 204.23 | 205.23 | 207.24 | 241 | 307.58 | 309.59 | ||||||
| 178 | 211.08 | 215.09 | 242 | 338.34 | 340.35 | ||||||||
| 179 | 223.11 | 227.13 | 243 | 369.35 | 370.35 | 371.36 | |||||||
| 180 | 226.95 | 227.95 | 230.96 | 244 | 64.016 | 65.019 | |||||||
| 181 | 230.99 | 235 | 245 | 73.065 | 74.068 | ||||||||
| 182 | 235.97 | 239.98 | 246 | 74.06 | 75.063 | ||||||||
| 183 | 237 | 241.01 | 247 | 76.039 | 77.043 | ||||||||
| 184 | 237.97 | 241.99 | 248 | 84.081 | 85.084 | ||||||||
| 185 | 246 | 250.01 | 249 | 86.096 | 87.1 | ||||||||
| 186 | 251.94 | 255.95 | 250 | 87.044 | 88.047 | ||||||||
| 187 | 257.95 | 261.96 | 251 | 89.06 | 90.063 | ||||||||
| 188 | 273.92 | 277.94 | 252 | 90.977 | 91.98 |
TABLE 1. Potential labeled ions in positive ion mode
| No. | M | M+1 | M+2 | M+3 | M+4 | M+5 | No. | M | M+1 | M+2 | M+3 | M+4 | M+5 |
| 253 | 94.065 | 95.068 | 317 | 197.08 | 198.08 | ||||||||
| 254 | 96.042 | 97.045 | 318 | 200.24 | 201.24 | ||||||||
| 255 | 99.042 | 100.04 | 319 | 203.05 | 204.06 | ||||||||
| 256 | 102.09 | 103.09 | 320 | 204.03 | 205.03 | ||||||||
| 257 | 104.11 | 105.11 | 321 | 204.12 | 205.13 | ||||||||
| 258 | 105.04 | 106.05 | 322 | 205.07 | 206.07 | ||||||||
| 259 | 107.05 | 108.06 | 323 | 205.1 | 206.1 | ||||||||
| 260 | 109.03 | 110.03 | 324 | 206.06 | 207.06 | ||||||||
| 261 | 113.06 | 114.06 | 325 | 209.11 | 210.12 | ||||||||
| 262 | 113.1 | 114.1 | 326 | 214.25 | 215.26 | ||||||||
| 263 | 115.04 | 116.04 | 327 | 216.92 | 217.93 | ||||||||
| 264 | 115.11 | 116.12 | 328 | 217.08 | 218.09 | ||||||||
| 265 | 117.09 | 118.09 | 329 | 217.1 | 218.11 | ||||||||
| 266 | 118.09 | 119.09 | 330 | 217.16 | 218.16 | ||||||||
| 267 | 120.07 | 121.07 | 331 | 218.14 | 219.14 | ||||||||
| 268 | 121.06 | 122.07 | 332 | 219.03 | 220.03 | ||||||||
| 269 | 123.04 | 124.05 | 333 | 226.01 | 227.02 | ||||||||
| 270 | 124.05 | 125.05 | 334 | 228.27 | 229.27 | ||||||||
| 271 | 128.02 | 129.02 | 335 | 230.25 | 231.25 | ||||||||
| 272 | 129.05 | 130.06 | 336 | 233.08 | 234.08 | ||||||||
| 273 | 129.09 | 130.09 | 337 | 236.07 | 237.07 | ||||||||
| 274 | 129.13 | 130.13 | 338 | 236.11 | 237.12 | ||||||||
| 275 | 130.16 | 131.16 | 339 | 237.15 | 238.15 | ||||||||
| 276 | 132.1 | 133.11 | 340 | 239.16 | 240.16 | ||||||||
| 277 | 133.09 | 134.09 | 341 | 241.07 | 242.07 | ||||||||
| 278 | 135.1 | 136.1 | 342 | 242.1 | 243.1 | ||||||||
| 279 | 136.02 | 137.02 | 343 | 242.28 | 243.29 | ||||||||
| 280 | 137.07 | 138.07 | 344 | 245.08 | 246.08 | ||||||||
| 281 | 139.05 | 140.05 | 345 | 253.14 | 254.14 | ||||||||
| 282 | 139.07 | 140.08 | 346 | 257.04 | 258.05 | ||||||||
| 283 | 139.11 | 140.12 | 347 | 257.15 | 258.15 | ||||||||
| 284 | 140.07 | 141.07 | 348 | 261.13 | 262.13 | ||||||||
| 285 | 141.09 | 142.09 | 349 | 265.11 | 266.11 | ||||||||
| 286 | 142.05 | 143.05 | 350 | 267.16 | 268.16 | ||||||||
| 287 | 143.11 | 144.11 | 351 | 274.27 | 275.28 | ||||||||
| 288 | 145.03 | 146.03 | 352 | 277.05 | 278.05 | ||||||||
| 289 | 145.12 | 146.13 | 353 | 277.1 | 278.11 | ||||||||
| 290 | 147.11 | 148.12 | 354 | 280.09 | 281.1 | ||||||||
| 291 | 148 | 149.01 | 355 | 284.33 | 285.33 | ||||||||
| 292 | 149.09 | 150.1 | 356 | 293.02 | 294.02 | ||||||||
| 293 | 150.06 | 151.06 | 357 | 293.24 | 294.25 | ||||||||
| 294 | 154.08 | 155.09 | 358 | 296.07 | 297.07 | ||||||||
| 295 | 155.07 | 156.07 | 359 | 301.14 | 302.14 | ||||||||
| 296 | 155.11 | 156.11 | 360 | 305.16 | 306.16 | ||||||||
| 297 | 157.08 | 158.09 | 361 | 309.2 | 310.21 | ||||||||
| 298 | 157.12 | 158.13 | 362 | 313.11 | 314.11 | ||||||||
| 299 | 158.96 | 159.97 | 363 | 317.11 | 318.12 | ||||||||
| 300 | 159.07 | 160.07 | 364 | 332.33 | 333.33 | ||||||||
| 301 | 161.09 | 162.1 | 365 | 335.01 | 336.01 | ||||||||
| 302 | 162.11 | 163.12 | 366 | 359.11 | 360.12 | ||||||||
| 303 | 166.09 | 167.09 | 367 | 360.32 | 361.33 | ||||||||
| 304 | 167.1 | 168.11 | 368 | 368.42 | 369.43 | ||||||||
| 305 | 169.05 | 170.05 | 369 | 370.35 | 371.36 | ||||||||
| 306 | 169.12 | 170.13 | 370 | 376.3 | 377.3 | ||||||||
| 307 | 173.06 | 174.06 | 371 | 398.24 | 399.24 | ||||||||
| 308 | 173.08 | 174.08 | 372 | 413.27 | 414.27 | ||||||||
| 309 | 177.09 | 178.09 | 373 | 424.28 | 425.29 | ||||||||
| 310 | 179.1 | 180.11 | 374 | 425.14 | 426.14 | ||||||||
| 311 | 182.08 | 183.08 | 375 | 429.24 | 430.24 | ||||||||
| 312 | 183.06 | 184.07 | 376 | 461.31 | 462.32 | ||||||||
| 313 | 183.14 | 184.14 | 377 | 475.33 | 476.33 | ||||||||
| 314 | 185.11 | 186.12 | 378 | 487.2 | 488.2 | ||||||||
| 315 | 189.05 | 190.06 | 379 | 507.27 | 508.27 | ||||||||
| 316 | 191.1 | 192.11 | 380 | 523.25 | 524.25 |
TABLE 2. Potential labeled ions in negative ion mode
| No. | M | M+1 | M+2 | M+3 | M+4 | M+5 | No. | M | M+1 | M+2 | M+3 | M+4 | M+5 |
| 1 | 114.056 | 119.0729 | 63 | 146.0652 | 149.0753 | 150.0787 | |||||||
| 2 | 128.0354 | 133.0521 | 64 | 147.0492 | 148.0527 | 149.056 | 150.0593 | 151.0628 | |||||
| 3 | 129.0194 | 134.0364 | 65 | 148.0333 | 149.0367 | 150.04 | 151.0433 | 152.0467 | |||||
| 4 | 129.0306 | 134.0469 | 66 | 156.9908 | 157.9942 | 161.0037 | |||||||
| 5 | 145.0143 | 150.031 | 67 | 173.0089 | 177.0225 | ||||||||
| 6 | 145.0619 | 149.0753 | 150.0787 | 68 | 174.0408 | 178.0543 | |||||||
| 7 | 146.0459 | 147.0493 | 148.0527 | 149.056 | 150.0593 | 151.0628 | 69 | 189.989 | 194.0023 | ||||
| 8 | 147.03 | 148.0333 | 149.0367 | 150.04 | 151.0433 | 152.0467 | 70 | 191.0198 | 193.0266 | 195.0328 | |||
| 9 | 149.0341 | 154.051 | 71 | 200.0176 | 204.031 | ||||||||
| 10 | 168.0278 | 173.0448 | 72 | 205.0077 | 207.0145 | 209.0211 | |||||||
| 11 | 169.0118 | 172.0218 | 174.0288 | 73 | 211.9707 | 215.984 | |||||||
| 12 | 181.0379 | 186.0553 | 74 | 212.8869 | 216.9002 | ||||||||
| 13 | 183.0356 | 188.0525 | 75 | 221.9996 | 226.0131 | ||||||||
| 14 | 188.9862 | 194.0023 | 76 | 247.9474 | 251.9609 | ||||||||
| 15 | 201.9968 | 206.0106 | 207.0145 | 77 | 263.9793 | 267.9931 | |||||||
| 16 | 203.0203 | 208.0372 | 78 | 329.0615 | 331.0683 | 333.075 | |||||||
| 17 | 204.0045 | 207.0145 | 209.0211 | 79 | 118.0227 | 119.026 | 121.0329 | ||||||
| 18 | 205.0175 | 210.0342 | 80 | 133.0336 | 135.0403 | 136.0437 | |||||||
| 19 | 205.992 | 207.9987 | 211.0086 | 81 | 134.0177 | 135.0211 | 136.0247 | 137.0277 | |||||
| 20 | 206.0016 | 211.0183 | 82 | 157.9942 | 161.0037 | ||||||||
| 21 | 206.9855 | 212.0024 | 83 | 192.0232 | 193.0266 | 195.0328 | |||||||
| 22 | 213.0493 | 215.0561 | 218.066 | 84 | 197.9631 | 200.9726 | |||||||
| 23 | 214.0333 | 217.0432 | 218.0466 | 219.0502 | 85 | 199.9214 | 202.931 | ||||||
| 24 | 215.0174 | 220.0339 | 86 | 204.9884 | 207.9987 | ||||||||
| 25 | 225.9865 | 228.9965 | 231.0032 | 87 | 215.056 | 218.066 | |||||||
| 26 | 226.9706 | 231.9873 | 88 | 319.9215 | 322.9316 | ||||||||
| 27 | 227.0652 | 232.0815 | 89 | 154.9187 | 156.9253 | ||||||||
| 28 | 227.9835 | 233.0002 | 90 | 172.0222 | 174.0288 | ||||||||
| 29 | 228.9675 | 233.9846 | 91 | 176.9359 | 178.943 | ||||||||
| 30 | 229.033 | 234.0498 | 92 | 102.9569 | 103.9603 | ||||||||
| 31 | 236.0152 | 241.032 | 93 | 104.9539 | 105.9573 | ||||||||
| 32 | 236.9993 | 242.0162 | 94 | 113.9934 | 114.9968 | ||||||||
| 33 | 250.0308 | 255.0476 | 95 | 116.9725 | 117.9758 | ||||||||
| 34 | 260.9801 | 265.9959 | 96 | 118.9695 | 119.9729 | ||||||||
| 35 | 261.963 | 266.9798 | 97 | 128.9596 | 129.9629 | ||||||||
| 36 | 262.976 | 263.9793 | 267.9931 | 98 | 130.0873 | 131.0908 | |||||||
| 37 | 263.96 | 268.9768 | 99 | 141.0169 | 142.0203 | ||||||||
| 38 | 266.0045 | 271.0214 | 100 | 152.996 | 153.9994 | ||||||||
| 39 | 271.0079 | 276.0247 | 101 | 171.0066 | 172.0099 | ||||||||
| 40 | 283.9449 | 288.9617 | 102 | 178.9727 | 179.9762 | ||||||||
| 41 | 284.9286 | 289.946 | 103 | 184.9599 | 185.9633 | ||||||||
| 42 | 285.942 | 290.9589 | 104 | 185.0222 | 186.0256 | ||||||||
| 43 | 293.9739 | 298.9906 | 105 | 188.9816 | 189.985 | ||||||||
| 44 | 306.0764 | 307.0798 | 308.0828 | 309.0866 | 310.0897 | 311.0933 | 106 | 194.9887 | 195.992 | ||||
| 45 | 307.0799 | 309.0866 | 310.0897 | 311.0933 | 312.0968 | 107 | 198.9756 | 199.979 | |||||
| 46 | 320.9345 | 325.9515 | 108 | 209.0043 | 210.0075 | ||||||||
| 47 | 328.0583 | 329.0618 | 331.0683 | 333.075 | 109 | 215.0328 | 216.036 | ||||||
| 48 | 364.035 | 369.052 | 110 | 218.1033 | 219.1068 | ||||||||
| 49 | 366.0315 | 371.0484 | 111 | 223.02 | 224.0234 | ||||||||
| 50 | 386.0168 | 391.0337 | 112 | 227.9448 | 228.948 | ||||||||
| 51 | 388.0138 | 393.0307 | 113 | 242.1762 | 243.1794 | ||||||||
| 52 | 443.9754 | 448.9926 | 114 | 252.9473 | 253.9507 | ||||||||
| 53 | 102.0561 | 106.0696 | 115 | 276.9916 | 277.9951 | ||||||||
| 54 | 103.0401 | 107.0536 | 116 | 291.0074 | 292.0108 | ||||||||
| 55 | 115.0037 | 119.0172 | 117 | 297.1528 | 298.1562 | ||||||||
| 56 | 117.0194 | 118.0227 | 119.026 | 121.0329 | 118 | 311.1686 | 312.1719 | ||||||
| 57 | 123.9986 | 128.0121 | 119 | 325.1841 | 326.1874 | ||||||||
| 58 | 129.0387 | 133.0521 | 120 | 339.1996 | 340.203 | ||||||||
| 59 | 130.0228 | 134.0364 | 121 | 372.3035 | 373.307 | ||||||||
| 60 | 132.0302 | 133.0336 | 135.0403 | 136.0437 | 122 | 382.3323 | 383.3357 | ||||||
| 61 | 133.0143 | 134.0177 | 135.0211 | 136.0247 | 137.0277 | 123 | 396.3478 | 397.3513 | |||||
| 62 | 142.9752 | 143.9787 | 146.988 |
TABLE 3 labeled ions and their secondary mass spectral information in positive ion mode
TABLE 3. Marked ions and their secondary mass spectral information under positive ion mode
TABLE 3. Marked ions and their secondary mass spectral information under positive ion mode
| Number of | Name of Compound | Number of marks | Number of precise mass | Second level information (relative intensity) |
| 39 | Unknown_p32 | M+0 | 198.0372 | 198.0367(1) |
| 39 | Unknown_p32 | M+4 | 202.0506 | 202.0505(1) |
| 40 | Unknown_p33 | M+0 | 199.9904 | 62.9817(0.51),199.9904(1) |
| 40 | Unknown_p33 | M+4 | 204.0039 | 62.9817(0.3),204.004(1) |
| 41 | Unknown_p34 | M+0 | 223.1111 | 223.1117(1) |
| 41 | Unknown_p34 | M+4 | 227.1252 | 227.1251(1) |
| 42 | Unknown_p35 | M+0 | 236.9979 | 132.9871(1.81),132.9871(1.81),236.998(1) |
| 42 | Unknown_p35 | M+4 | 241.0113 | 134.9938(0.82),135.9971(0.78),241.0111(1) |
| 43 | Unknown_p36 | M+0 | 257.949 | 62.9817(0.61),155.0271(1.69),183.0222(2.1),199.9904(5.59),257.9487(1) |
| 43 | Unknown_p36 | M+4 | 261.9623 | 62.9817(0.32),159.0406(1.42),187.0354(0.89),204.0039(3.94),261.9621(1) |
| 44 | Unknown_p37 | M+0 | 377.0815 | 297.0884(0.3),377.0817(1) |
| 44 | Unknown_p37 | M+2 | 379.088 | 299.0951(0.32),379.088(1) |
| 45 | Unknown_p38 | M+0 | 88.0392 | 88.0391(1) |
| 45 | Unknown_p38 | M+3 | 91.0493 | 91.0492(1) |
| 46 | Unknown_p39 | M+0 | 214.0061 | 214.006(1) |
| 46 | Unknown_p39 | M+3 | 217.0164 | 217.0159(1) |
| 47 | Unknown_p40 | M+0 | 214.9901 | 62.9818(0.85),214.99(1) |
| 47 | Unknown_p40 | M+3 | 218.0004 | 62.9818(0.91),218.0002(1) |
| 48 | Unknown_p41 | M+0 | 215.016 | 215.0158(1) |
| 48 | Unknown_p41 | M+2 | 217.0229 | 217.0222(1) |
| 49 | Unknown_p42 | M+0 | 369.3512 | 369.3519(1) |
| 49 | Unknown_p42 | M+2 | 371.3579 | 371.3576(1) |
| 50 | Succinate semialdehyde | M+0 | 103.0388 | 70.9795(1) |
| 50 | Succinate semialdehyde | M+4 | 107.0522 | 70.9795(1) |
| 51 | Ornithine | M+0 | 133.097 | 74.0235(1) |
| 51 | Ornithine | M+5 | 138.1139 | 76.0302(1) |
| 52 | Putrescine | M+0 | 89.1072 | 72.0806(1) |
| 52 | Putrescine | M+4 | 93.1207 | 76.094(1) |
| 53 | Asparagine | M+0 | 133.0607 | 74.0235(1) |
| 53 | Asparagine | M+4 | 137.074 | 76.0302(1) |
| 54 | Sperimidine | M+0 | 146.1651 | 72.0806(1) |
| 54 | Sperimidine | M+4 | 150.1785 | 76.094(1) |
| 55 | Alanine | M+0 | 90.0549 | 73.0839(1) |
| 55 | Alanine | M+3 | 93.0649 | 76.094(1) |
TABLE 4. Labeled ions and their secondary mass spectral information in anion mode
TABLE 4. Marked ions and their second mass spectral information under negative ion mode
The analysis method can carry out quantitative analysis on the stable isotope labeled metabolites in a small amount of cells, and the analysis time is greatly shortened by direct sample injection. The method has the advantages of simple pretreatment, economy, high efficiency, high sensitivity and high flux.
Claims (1)
1. A stable isotope labeling metabolic flow analysis method based on chip nanoliter electrospray mass spectrometry is characterized by comprising the following steps:
(1) Directly extracting metabolites from cells by adopting methanol/water; the cells are cultured in two culture media respectively, one is a stable isotope labeling culture medium, and the other is an unstable isotope labeling culture medium; obtaining two cell metabolites which contain stable isotope labeling metabolites and contain metabolites which are not labeled by the stable isotope;
(2) Transferring the two cell metabolites into a sample injection disc respectively, adding a spray solvent, and directly injecting the mixture into a mass spectrometer through a chip-based multichannel nanoliter electrospray ion source;
(3) Acquiring primary mass spectrum information and secondary mass spectrum information by adopting a full-scanning and parallel reaction monitoring mode, and finding out a metabolite marked by a stable isotope through primary mass spectrum information matching and secondary mass spectrum information matching;
(4) Quantifying the metabolite marked by the stable isotope in the sample by adopting a parallel reaction monitoring mode;
the cells in the step (1) are respectively cultured in two culture media, one is a stable isotope labeling culture medium which contains stable isotope labeling metabolites 13 C 6 -a glucose, 13 c-1,2-glucose, 13 C 3 -pyruvic acid, 13 C 5 -a source of glutamine, 15 N 2 -a source of glutamine, 13 C 5 - 15 N 2 -a source of glutamine, 2 H 3 -one or more than two of serine; the other is an unstable isotope labeled culture medium, which contains metabolites that are not labeled with stable isotopes, and adopts unstable isotope labeling corresponding to stable isotope labeling and stable isotope labeling in concentration and stable isotope labeled culture mediumIsotopically labeled metabolites are consistent; culturing cells in 6-well, 12-well, 24-well, or 96-well or 10cm culture plates to make the cell density of each well reach 50% -90%; discarding the culture medium and washing twice with PBS; counting the cells, adding 50-100 mu L of 60-80% methanol/water (v/v) extracting agent into each 10000-20000 cells, wherein the extracting agent contains methionine sulfone and camphorsulfonic acid sodium salt with final concentration of 5-20 mu M respectively as internal standard; covering with aluminum foil paper and sealing film, standing at 3-5 deg.C for 30-40min to extract metabolite; transferring 20 to 40 mu L of the extracting solution into a sample plate in the step (2), and adding a spray solvent with the volume equal to that of the extracting solution into each hole, namely 20 to 40 mu L of the spray solvent; directly injecting the nano-liter electrospray ion source into a mass spectrometer through a chip-based multi-channel nano-liter electrospray ion source;
under the positive ion detection mode, the spraying solvent is 60-80% methanol/water (v/v) and contains 0.3-0.5% formic acid (v/v); under the negative ion detection mode, the spraying solvent is 60-80% methanol/water (v/v), ammonium acetate containing 10-30mM and 0.03-0.05% formic acid (v/v);
performing mass spectrometry on the cell extracting solution cultured in the stable isotope labeling culture medium and the cell extracting solution cultured in the non-stable isotope labeling culture medium by adopting a full scanning and parallel reaction monitoring mode in the step (3), and finding out the metabolite labeled by the stable isotope through matching of a primary mass spectrum and a secondary mass spectrum;
the specific process is as follows:
(1) First-order mass spectrum matching, finding potential labeled metabolites: analyzing a cell extracting solution cultured in a stable isotope labeling culture medium and a cell extracting solution cultured in an unstable isotope labeling culture medium by adopting a full scanning mode; filtering by primary mass spectrometry to obtain characteristic ions which are not marked and stably exist in a marked cell extracting solution, wherein the signal-to-noise ratio is less than 3, less than 60-80% of metabolites appear in repeated samples for more than 3 times, and the metabolites existing in blank samples are filtered; wherein the blank sample refers to a blank solvent without containing a cell sample, namely a mixed solution of an extracting agent and a spraying solvent;
calculating the accurate mass number of the corresponding isotope on the label as M + n according to the accurate mass number of the characteristic ions stably existing in the unlabeled sample as M, wherein n is the number of atoms marked by the stable isotope and is matched with the characteristic ions stably existing in the labeled cell extracting solution; the matching rules are equal under the mass deviation of 5-15 ppm; if there is a match, the metabolite is considered to be a potential labeled metabolite; if not, neglecting;
(2) Matching the secondary mass spectrum, and confirming the labeled metabolite information; aiming at potential labeled metabolites, a parallel reaction monitoring mode is adopted to obtain secondary mass spectrum information;
extracting secondary mass spectrograms with accurate mass numbers M and M + n from a stable isotope labeled sample respectively, generating all secondary ion primary and secondary ions → daughter ions according to M, abbreviated as M → Mp, calculating the accurate mass number of the stable isotope labeled secondary ions, and matching with the secondary mass spectrogram of M + n, wherein the secondary mass spectrogram of M + n is the primary ions → the daughter ions, abbreviated as M + n → Mp + M, M refers to the number of atoms labeled by the stable isotope, and M is less than or equal to n; the matching rules are equal under the mass deviation of 5-20 ppm; if not, neglecting; if the values are matched, calculating the ratio of the Mp + m intensity to the Mp intensity; meanwhile, extracting M → Mp and M + n → Mp + M from a sample without stable isotope labeling, calculating the ratio of the strength of Mp + M to the strength of Mp, and if the value obtained by multiplying the ratio by 1.0 to 1.2 is smaller than the ratio of the strength of Mp + M to the strength of Mp in the sample with stable isotope labeling, considering that M and M + n are a pair of non-labeled and labeled metabolites and respectively contain secondary fragments Mp and Mp + M;
according to the above rules, metabolites labeled by stable isotopes can be found, and the primary and secondary mass spectrum information thereof can be used for qualitative and quantitative analysis;
quantifying the metabolite marked by the stable isotope by adopting a parallel reaction monitoring mode in the step (4);
the conditions for the parallel reaction monitoring mode were: the maximum ion collection time is 100 to 200ms, each 0.1 to 0.3s is a time period, and no more than 50 ions are contained in one time period so as to ensure that each ion is collected at least to more than 5 points;
meanwhile, the internal standard intensity is collected in the whole section; for each ion, the intensity was divided by the internal standard intensity at the same time, and the average of 5 or more points was taken as a semi-quantitative result.
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| JP2006337176A (en) * | 2005-06-02 | 2006-12-14 | Eisai R & D Management Co Ltd | Determination method of metabolite |
| WO2018174891A1 (en) * | 2017-03-23 | 2018-09-27 | Mprobe Inc. | Quantitative targeted metabolomic analysis based on the mixture of isotope-and nonisotope-labeled internal standards |
| JP2019024326A (en) * | 2017-07-25 | 2019-02-21 | 大陽日酸株式会社 | Method of producing labelled metabolite, method of quantifying metabolite, and labelled metabolite production kit |
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| US8510054B2 (en) * | 2004-02-05 | 2013-08-13 | Ajinomoto Co., Inc. | Intracellular metabolic flux analysis method using substrate labeled with isotope |
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| JP2006337176A (en) * | 2005-06-02 | 2006-12-14 | Eisai R & D Management Co Ltd | Determination method of metabolite |
| WO2018174891A1 (en) * | 2017-03-23 | 2018-09-27 | Mprobe Inc. | Quantitative targeted metabolomic analysis based on the mixture of isotope-and nonisotope-labeled internal standards |
| JP2019024326A (en) * | 2017-07-25 | 2019-02-21 | 大陽日酸株式会社 | Method of producing labelled metabolite, method of quantifying metabolite, and labelled metabolite production kit |
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