CN112540139B - Molecular weight calibrator kit for metabolic profiling detection, and preparation method and use method thereof - Google Patents
Molecular weight calibrator kit for metabolic profiling detection, and preparation method and use method thereof Download PDFInfo
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Abstract
The invention relates to a molecular weight calibrator kit for metabolic spectrum detection and a preparation method and a using method thereof, wherein the kit comprises a metabolic molecular calibrator, a dissolved solution and a molecular weight calibration list; wherein, the proportion of the metabolic molecule calibrator and the dissolving solution is as follows: 1 part of metabolic molecule calibrator corresponds to 1-5 parts of dissolving solution. The molecular weight calibrator is taken from a biological sample, so that the cost is low and the preparation is convenient; the device can cover the 0-1100Da range of metabolic molecule detection, is simultaneously suitable for a positive ion detection mode and a negative ion detection mode, and can select a molecular weight calibrator and a calibration list according to needs; the method is convenient to use, and can be suitable for detection and molecular weight calibration of metabolic molecular regions of various ion source mass spectrometers and various biological samples; the invention is suitable for accurate molecular weight determination of the metabolic small molecules, structure identification and analysis of the metabolic small molecules and molecular accurate mass spectrum imaging.
Description
Technical Field
The invention relates to the technical field of mass spectrometry, in particular to a molecular weight calibrator kit for metabolic spectrum detection, and a preparation method and a use method thereof.
Background
The metabolic molecules can reflect the phenotypic change, physiological condition and pathological condition of organisms in real time, and the composition change of the metabolic molecules in complex biological samples such as blood, saliva, urine, tissues, cells and the like is closely related to disease monitoring. MALDI-MS matrix assisted laser desorption ionization mass spectrometry is a high-flux mass spectrometry technology, which is widely applied to detection and analysis of macromolecules such as protein, polypeptide, nucleic acid and the like, and has attracted great attention in the detection of metabolic small molecules in recent years. However, because the composition of the metabolic molecules is various, taking lipid as an example, more than 20000 species have been discovered and named, and when MALDI and other direct ionization mass spectrometry technologies are used as platforms to perform direct detection and analysis of complex biological samples, the molecular weight calibration is very important, and the reliability and accuracy of the metabolic spectrogram are directly influenced. The composition of metabolic molecules in a biological sample is complex, and part of metabolic molecules appear in the form of cluster peaks, and the detection range of a direct ionization mass spectrometer such as MALDI is mainly concentrated in the range of 50-1200 Da. At present, no kit or product of the small molecular weight calibrator capable of fully covering the range exists.
Commercial lipid calibrators developed by Avanti
Can cover 400-900Da, but is formed by mixing a plurality of pure lipid products, has higher cost and high price, and is not easy to store because of being dissolved in methanol solution and being volatile. Organic acids commonly used in MALDI such as CHCA, DHB, SA and the like can also be used as molecular weight calibrators of small molecules, but highly nonuniform crystals are easily formed, so that the molecular weight precision during mass spectrum detection is influenced, a proper calibration effect cannot be achieved, and the molecular weight of cluster peaks formed by the crystals is analyzed within a range of 50-500Da, which is insufficient for detection of metabolic molecules in a high molecular region.
The patent CN104597114B proposes a mixture of fatty acids as a molecular weight calibrator for the metabolite region in the negative ion detection mode, but only calibrates the range of 100-350 Da. According to the calibration principle, if the molecular weight of the calibrator is not uniformly distributed in the required detection range, the calibration is prone to serious deviation.
Patent CN109632938A utilizes AgNO3The silver cluster peak in the @ PDA nano-particle is used as a molecular weight calibrator, and the metabolism molecules are spotted on AgNO3The surface of the @ PDA nano-particle is provided with silver clusters which become an internal standard calibrator under the positive ion detection mode. The method can cover the molecular weight range of 300-1000Da, but the silver cluster peak can seriously interfere the spectrogram of a complex biological sample to obtain the peak, and simultaneously, the ion addition form of the metabolic molecules, the spectrogram form of the metabolic molecules and the distribution condition of nanoparticles on a substrate are changedIn correlation, the stability of the spectrogram cannot be guaranteed. And the silver clusters form a molecule list capable of calculating accurate molecular weight only in the positive ion mode, and cannot be simultaneously used for calibrating the metabolic molecule region in the negative ion mode. Meanwhile, in the direct ionization mass spectrometry detection technology, the ion suppression effect cannot be avoided, and other internal standard methods are not beneficial to the accurate detection of complex biological samples.
The market is in need of developing a molecular weight calibration kit for metabolic molecules, which has low cost, convenient use and wide coverage range.
Disclosure of Invention
The invention aims to provide a molecular weight calibrator kit for metabolic spectrum detection in order to overcome the defects of the prior art, the preparation method of the kit is simple, the formula and the molecular weight calibration list of the kit can be adjusted according to the calibration range, the negative ion mode and the positive ion mode are both considered, and the defect that different detection modes are respectively calibrated is avoided; the calibration precision is high; the kit can be suitable for mass spectrometry detection application scenes of complex biological samples such as tissues, serum, plasma, bile, cerebrospinal fluid, urine, saliva and the like.
Another purpose of the invention is to provide a preparation method of the kit.
The invention also aims to provide a using method of the kit.
In order to achieve the purpose, the invention adopts the following technical scheme:
a molecular weight calibrator kit for metabolic profiling detection, the kit comprising a metabolic molecular calibrator, a dissolution solution and a molecular weight calibration list; wherein, the proportion of the metabolic molecule calibrator and the dissolving solution is as follows: 1 part of metabolic molecule calibrator corresponds to 1-5 parts of dissolving solution.
In the above molecular weight calibrator kit for metabolic profiling detection, the metabolic molecular calibrator is extracted from a biological sample, and the biological sample includes: mammalian serum, plasma, milk, bile, cerebrospinal fluid, saliva, urine, sweat, cells, tissue, cell lysate, tissue extract, bacterial lysate, or fungal lysate.
The molecular weight calibrator kit for metabolic profiling detection comprises the following dissolving solution: water, methanol, ethanol, acetonitrile, isopropanol, acetone, and a mixed solvent of two or more thereof.
In the molecular weight calibrator kit for metabolic profiling detection, the molecular weight calibration list of the biological sample as a tissue sample includes: mass-to-charge ratio m/z in negative ion mode is 255.2330, 281.2486, 303.2330, 419.2568, 599.3202, 806.5458, 885.5499, 878.6033, 888.6240, 906.6346, 1042.6717 and 1070.7030; in positive ion mode, the mass to charge ratio m/z is 184.1097, 478.2330, 496.3398, 524.3711, 734.5694, 760.5851, 782.5676, 798.5410, 806.5670, 832.5832, 848.5566, 869.7000, 923.7470 and 949.7621.
In the foregoing molecular weight calibrator kit for metabolic profiling, a molecular weight calibration list of a biological sample that is a serum sample includes: the mass-to-charge ratio m/z in the negative ion mode is 152.9699, 255.2330, 279.2330, 283.2643, 303.2330, 367.1551, 435.2482, 465.3030, 616.4784, 642.4990, 687.5554, 726.6026, 752.6139, 778.5289, 794.5272, 833.5268, 861.5499, 885.5499, 904.6189 and 922.6131, and the range of 100-1000Da can be calibrated; the mass-to-charge ratio m/z in positive ion mode is 184.1097, 437.1032, 459.1522, 478.2330, 502.2330, 546.2744, 635.1439, 659.2695, 678.5991, 690.4992, 725.5410, 780.5514, 808.5560 and 832.5827, and the range of 150-900Da can be calibrated.
In the molecular weight calibrator kit for metabolic profiling detection, the molecular weight calibration list of the saliva and urine samples as the biological samples comprises: the mass/charge ratio m/z in the negative ion mode is 57.97152, 61.9812, 84.98303, 89.0226, 96.9641, 119.9455, 124.0074, 135.9187, 146.9659, 167.0217, 219.1761, 255.2328, 265.1451 and 311.1662, and the range of 0-350Da can be calibrated; the mass/charge ratio m/z in positive ion mode is 74.2500, 91.0540, 103.9910, 170.0970, 238.3760, and can be calibrated in the range of 0-300 Da.
The preparation method of the molecular weight calibrator kit for metabolic profiling detection comprises the following steps:
step one, preparing a metabolic molecule calibrator: extracting metabolic molecules in different biological samples by using an extraction reagent to obtain a calibrator containing the metabolic molecules;
step two, measuring the molecular weight of the metabolic molecules in the metabolic molecule calibrator:
1) mixing the dissolved solution and the metabolic molecule calibrator to obtain a metabolic molecule calibrator solution, and adding a pure calibrator serving as an internal standard into the metabolic molecule calibrator solution to prepare a metabolic molecule calibrator solution containing the internal standard;
2) spotting the metabolic molecule calibrator solution containing the internal standard on a sample loading platform, and naturally drying;
3) putting the sample carrying table into a mass spectrometer for detecting a positive ion mode and a negative ion mode, and determining the molecular weight of each mass spectrum peak in the metabolic molecule calibrator solution under the two detection modes by using an internal standard method;
4) selecting a mass spectrum peak with high peak intensity and stability to perform secondary mass spectrum detection, obtaining a secondary mass spectrum, and performing structure identification on the secondary mass spectrum to obtain theoretically accurate molecular weight;
5) detecting the metabolic molecule calibrator containing the internal standard again, determining and correcting the accurate molecular weight of the peak of the metabolic molecule calibrator under two detection modes except the known theoretical molecular weight by taking the pure calibrator and the peak with the definite structure and the theoretical accurate molecular weight as the internal standard, and obtaining an accurate molecular weight list;
6) selecting high-intensity and stable peaks in the accurate molecular weight list, and establishing a molecular weight calibration list in mass spectrometer control software;
step three, preparing the kit according to the following formula: the formula comprises the following components: a metabolic molecule calibrator, a dissolving solution and a molecular weight calibration list; the proportion of the metabolic molecule calibrator to the dissolving solution is as follows: 1 part of metabolic molecule calibrator corresponds to 1-5 parts of the dissolution solution.
Determining accurate molecular weight lists of the metabolic molecule calibrator in two detection modes by combining an internal standard method and secondary mass spectrum identification; wherein the list of accurate molecular weights comprises: in negative ion mode, m/z is 57.97152, 61.9812, 84.98303, 89.0226, 96.9641, 119.9455, 124.0074, 135.9187, 146.9659, 152.9699, 167.0217, 219.1761, 255.2330, 265.1451, 279.2330, 281.2486, 283.2643, 303.2330, 367.1551, 419.2568, 435.2482, 465.3030, 599.3202, 616.4784, 642.4990, 687.5554, and 687.5554.
The mass spectrum peaks of the pure calibrator detected in the negative ion mode include: 89.0244, 124.0074, 167.0211, 199.1704, 255.2330, 283.2643, 311.2956, 319.1664, 367.3582, 690.5079, and the mass spectrum peaks of the pure calibrator detected in positive ion mode include: m/z is 91.0548, 170.0970, 184.1097, 790.6326, 812.6145 and 829.7256.
The application method of the molecular weight calibrator kit for detecting the metabolic spectrum comprises an internal standard calibration method and an external standard calibration method; the internal standard calibration method is used for metabolic molecules overlapped with the metabolic molecule calibrator in the sample to be tested, and the external standard calibration method does not stipulate the components of the sample to be tested.
In the application method of the molecular weight calibrator kit for metabolic profiling detection, the internal standard calibration method comprises the following steps:
step one, calibrating the molecular weight of a biological sample to be tested:
1) obtaining metabolic molecules in a biological sample to be detected;
2) dissolving 1 part of metabolic molecule calibrator by using a dissolving solution to obtain a metabolic molecule calibrator solution;
3) transferring the metabolic molecules in the biological sample to be detected to a sample carrying table, and simultaneously spotting the metabolic molecule calibrator solution on the same sample carrying table;
4) starting a mass spectrometer to collect data, calibrating the spectrometer in two detection modes by using a metabolic molecule calibrator and a molecular weight calibration list, and storing the method;
5) after the calibration of the instrument is completed, performing spectrogram acquisition on a sample to be detected, confirming a molecular weight calibration list capable of stably generating peaks in the sample to be detected, eliminating peaks which cannot stably generate peaks in the sample to be detected in the molecular weight calibration list of the kit, and performing internal standard calibration on the sample to be detected in each hole by using the modified molecular weight calibration list;
step two, evaluating the calibration effect:
1) performing secondary identification on the peak with higher peak intensity and more stable peak in the spectrogram of the sample to be detected, and confirming that the peak and the corresponding peak in the molecular weight calibration list are from the same molecule;
2) comparing the mass-to-charge ratio of the calibrated peaks of the internal molecular weight standards at multiple sites with the accurate molecular weights in the molecular weight calibration list, and calculating the calibration accuracy (ppm) by the formula of | m/z(molecular weight after calibration)-m/z(accurate molecular weight)|/m/z(accurate molecular weight)×106For evaluating the molecular weight calibration effect;
the use method of the internal standard calibration method in the data analysis software comprises the following steps:
step one, acquiring a metabolic spectrogram of a sample:
1) collecting, extracting or enriching metabolic molecules in a biological sample;
2) transferring the metabolic sample collected, extracted or enriched in the biological sample to a sample loading platform, and starting a mass spectrometer to collect data;
step two, realizing internal standard calibration in data analysis software:
transferring the metabolic spectrogram acquired by the mass spectrometer into data analysis software, calling a molecular weight calibration list, matching standard molecular weights in the molecular weight calibration list with detected molecular weights in a sample mass spectrogram, fitting a calibration curve according to a linear equation or a quadratic equation, and applying the calibration curve to each metabolic spectrogram to realize calibration of a full spectrogram;
step three, evaluating the calibration effect:
from a plurality of sample spectraThe mass-to-charge ratio of the peak of the molecule after calibration with the molecular weight internal standard is compared with the accurate molecular weight in the molecular weight calibration list, and the relative error (ppm) is calculated by the formula of | m/z(molecular weight after calibration)-m/z(accurate molecular weight)|/m/z(accurate molecular weight)×106And is used for judging the molecular weight calibration effect.
The method for using the molecular weight calibrator kit for metabolic profiling detection comprises the following steps: instrument detection control software; the use method of the external standard calibration method comprises the following steps:
step one, calibrating the molecular weight of a biological sample to be tested:
1) collecting, extracting or enriching metabolic molecules in a biological sample;
2) dissolving 1 part of metabolic molecule calibrator by using a dissolving solution to obtain a metabolic molecule calibrator solution;
3) transferring the metabolic sample collected, extracted or enriched in the biological sample to a sample loading platform, and simultaneously applying the sample of the metabolic calibrator solution to the sample loading platform, wherein the sample points to be detected are distributed around or adjacent to the metabolic molecule calibrator points;
4) starting a mass spectrometer to acquire data, performing external standard calibration on the molecular weight of the mass spectrometer in a negative ion mode and a positive ion mode by using a metabolic molecule calibrator and a molecular weight calibration list, and performing spectrogram acquisition on a sample to be detected in a sample hole to be detected in two detection modes after calibration;
step two, evaluating the calibration effect:
1) performing secondary identification on a peak with higher peak intensity and more stable peak in a spectrogram of a sample to be detected, wherein the secondary identification can confirm the peak of the molecular structure and calculate the theoretical molecular weight of the peak;
2) comparing the mass-to-charge ratio of the peaks after molecular weight calibration on multiple sites with the theoretically accurate molecular weight, and calculating the relative error (ppm) by using the formula of | m/z(molecular weight after calibration)-m/z(theoretical molecular weight)|/m/z(theoretical molecular weight)×106And is used for judging the molecular weight calibration effect.
The method for using the molecular weight calibrator kit for metabolic profiling detection comprises the following steps: a time-of-flight ion source mass spectrometer, a SIMS ion source mass spectrometer, or an atmospheric open ion source mass spectrometer; the sample loading platform of the mass spectrometer is a conductive substrate and comprises a stainless steel target plate or a nanostructure chip.
Compared with the prior art, the invention has the following beneficial effects:
1) the metabolic molecule calibrator in the molecular weight calibrator kit is taken from a biological sample, so that the cost is low and the preparation is convenient;
2) the molecular calibrator and the molecular weight external standard calibration list thereof can cover the 0-1100Da range of metabolic molecule detection, are simultaneously suitable for a positive ion detection mode and a negative ion detection mode, and can select the molecular weight calibrator and the calibration list as required;
3) the molecular weight calibrator kit is convenient to use, and is suitable for detection and molecular weight calibration of metabolic molecular regions of various ion source mass spectrometers and various biological samples;
4) the invention is suitable for accurate molecular weight determination of the metabolic small molecules, structural identification and analysis of the metabolic small molecules and molecular accurate mass spectrum imaging.
Drawings
FIG. 1 is a flow chart of the present invention;
FIG. 2 is a peak plot of a pure internal standard calibrator of the present invention mixed with a metabolic molecular calibrator in the negative ion mode;
FIG. 3 is a peak-appearing spectrum of the metabolite calibrator extracted from the serum according to the present invention in the negative ion mode;
FIG. 4 is a peak spectrum of a metabolite calibrator extracted from serum according to the present invention in positive ion mode;
FIG. 5 is a table of the calibration accuracy of the present invention after calibration with a molecular weight internal standard;
FIG. 6 is a peak profile of the metabolite calibrator extracted from the tissues of the present invention in the negative ion mode;
FIG. 7 is a peak profile of a metabolite calibrator extracted from a tissue according to the present invention in positive ion mode;
FIG. 8 is a graph of the accuracy of the calibration of the present invention using tissue-derived metabolic molecule calibrators as evaluation sites;
FIG. 9 is a graph showing the accuracy of the calibration of the present invention using a serum-derived metabolite calibrator as an evaluation site.
Detailed Description
Specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings, and it should be noted that the embodiments described herein are only for illustration and are not intended to limit the present invention. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, it will be understood by those of ordinary skill in the art that these specific details are not required in order to practice the present invention. Furthermore, in some embodiments, well-known circuits, materials, or methods have not been described in detail in order to avoid obscuring the present invention.
The invention provides a molecular weight calibrator kit for metabolic spectrum detection, which comprises a metabolic molecular calibrator, a dissolving solution and a molecular weight calibration list; wherein, the proportion of the metabolic molecule calibrator and the dissolving solution is as follows: 1 part of metabolic molecule calibrator corresponds to 1-5 parts of the dissolution solution.
In the present invention, the mass spectrometer includes: time-of-flight ion source mass spectrometer, SIMS ion source mass spectrometer, atmospheric open ion source mass spectrometer.
The sample loading platform comprises a stainless steel target plate, a nano material chip or other conductive substrates.
Example 1 extraction and preparation of Metabolic molecule calibrators from tissue samples
Step one, weighing 50mg of kidney, liver and brain tissues of a model animal, respectively adding 150 mu L of ice, and grinding until no obvious particles exist to obtain 3 tissue homogenate;
step two, transferring the 3 emulsions to reagent bottles respectively, adding 1mL of organic extraction solvent into the reagent bottles respectively, and performing oscillation incubation, centrifugation and washing to obtain metabolic molecule extraction phases of 3 tissues;
and step three, mixing the metabolic molecule extracts of the 3 tissues in equal volume, and evaporating the solvent to dryness to obtain the metabolic molecule calibrator.
And step four, re-dissolving the metabolic molecule calibrator with a dissolving solution to prepare a metabolic molecule calibrator solution. Preferably, the solution of the metabolic molecule calibrator extracted from the tissue sample is a mixed solvent of isopropanol and water, and the ratio of the metabolic molecule calibrator to the solution is 1: 5.
Example 2 extraction and preparation of Metabolic molecule calibrators from serum samples
Step one, obtaining a blood sample, coagulating and centrifuging to obtain clear light yellow serum supernatant;
step two, taking 10-50 mu L of serum supernatant, adding a mixed extraction solvent with the volume 5-10 times that of the serum supernatant, and carrying out oscillation incubation and centrifugation to layer a serum sample;
and step three, separating the serum layered sample obtained in the step two to obtain a solution containing the metabolic molecules, and evaporating the solvent of the solution to dryness to obtain the metabolic molecule calibrator.
And step four, re-dissolving the metabolic molecule calibrator with a dissolving solution to prepare a metabolic molecule calibrator solution. Preferably, the dissolving solution of the metabolic molecule calibrator extracted from the serum sample is a mixed solvent of isopropanol and water, and the ratio of the metabolic molecule calibrator to the dissolving solution is 1: 2.
Example 3 extraction and preparation of Metabolic molecule calibrators from saliva samples
Step one, obtaining a saliva sample, and removing mucin, cell debris and bacteria of the saliva sample through steps of filtering, centrifuging and the like to obtain clear transparent liquid;
step two, taking 10-50 mu L of clear and transparent saliva sample, adding 1-5 times of volume of extraction solvent, and performing oscillation incubation and centrifugation to precipitate protein, wherein the upper layer is solution containing metabolic molecules;
and step three, separating the sample in the step two to obtain a solution containing the metabolic molecules, and evaporating the solvent to dryness to obtain the metabolic molecule calibrator.
And step four, re-dissolving the metabolic molecule calibrator with a dissolving solution to prepare a metabolic molecule calibrator solution. Preferably, the dissolving solution of the metabolic molecule calibrator extracted from the saliva sample is a mixed solvent of methanol, acetonitrile and water, and the ratio of the metabolic molecule calibrator to the dissolving solution is 1: 2.
Example 4 extraction and preparation of a Metabolic molecule calibrator from urine samples
Step one, obtaining a urine sample, and removing cell debris, impurities and high-concentration salt through steps of filtering, dialyzing, centrifuging, diluting and the like to obtain clear and transparent liquid;
step two, taking 10-50 mu L of clear and transparent urine sample, adding 1-5 times of volume of extraction solvent, and performing oscillation incubation and centrifugation to precipitate protein, wherein the upper layer is solution containing metabolic molecules;
and step three, separating the sample in the step two to obtain a solution containing the metabolic molecules, and evaporating the solvent of the solution to dryness to obtain the metabolic molecule calibrator.
And step four, re-dissolving the metabolic molecule calibrator with a dissolving solution to prepare a metabolic molecule calibrator solution. Preferably, the dissolving solution of the metabolic molecule calibrator extracted from the urine sample is a mixed solvent of methanol, acetonitrile and water, and the ratio of the metabolic molecule calibrator to the dissolving solution is 1: 5.
FIG. 1 is a flow chart of the process of extracting and preparing a metabolite calibration sample from a biological sample.
It is worth noting that the samples used for the extraction of the metabolic molecular calibrator include, in addition to the tissue, serum, saliva and urine described in examples 1-4: human or mammalian plasma, milk, bile, cerebrospinal fluid, sweat, cells, tissues, cell lysates, tissue extracts, bacterial lysates, fungal lysates, etc.
Example 5 molecular weight determination of Metabolic molecular calibrator and obtaining molecular weight calibration List
Adding 100-1000 mu L of dissolving solution into a metabolic molecule calibrator, oscillating and redissolving to prepare a calibrator solution, subpackaging 10 mu L/tube by a single tube, and storing at-80 ℃ when not used;
adding pure mixed metabolic molecules consisting of lauric acid, palmitic acid, stearic acid, arachidic acid, lignoceric acid and 1, 2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine into the calibrator solution to form a metabolic molecule calibrator under an anion mode containing an internal standard;
adding pure mixed metabolic molecules consisting of 1, 2-distearoyl-sn-glycerol-3-phosphorylcholine and 1,2, 3-tripalmitin into the calibrator solution to form a metabolic molecule calibrator under a positive ion mode containing an internal standard;
and step four, spotting the metabolic molecule calibrator solution containing the internal standard on the nano mass spectrometry chip at 0.5-2 mu L/hole, and naturally drying the metabolic molecule calibrator solution at 10-20 sites of each point in the two detection modes. For the specific structure of the nano mass spectrometry chip, reference may be made to patents such as CN110530965A of this department;
putting the sample carrying table into a mass spectrometer for detection, determining the molecular weight of each mass spectrum peak in the metabolite calibrator solution under the detection mode of negative ions and positive ions by using an internal standard method, wherein the peak position of a pure mixed metabolite molecule consisting of lauric acid, palmitic acid, stearic acid, arachidic acid, lignoceric acid and 1, 2-dipalmitic acid-sn-glycerol-3-phosphoethanolamine under the negative ion mode is as follows: m/z 199.1704, 255.2330, 283.2643, 311.2956, 367.3582, 690.5079; the peak positions of pure mixed metabolism molecules consisting of 1, 2-distearoyl-sn-glycero-3-phosphocholine and 1,2, 3-tripalmitin in positive ion mode include: m/z 184.1097, 790.6326, 812.6145, 829.7256;
step six, selecting a mass spectrum peak with high peak intensity and stability to perform secondary mass spectrum detection, acquiring a secondary mass spectrum, and performing structure identification on the secondary mass spectrum to obtain theoretical molecular weight, wherein as an embodiment, when the metabolite calibrator is extracted from a tissue sample, the theoretical molecular weight of molecules with structures capable of being determined in a negative ion mode comprises m/z of 255.2330, 279.2330, 281.2486, 283.2643, 303.2330, 806.5458, 885.5499, 878.6033, 888.6240, 906.6346, 920.7397, 1042.6717 and 1070.7030; theoretical molecular weights of molecules that can determine structure in positive ion mode include m/z 184.1097, 478.2330, 496.3398, 524.3711, 734.5694, 760.5851, 782.5676, 798.5410, 806.5670, 832.5832, 848.5566, 853.7261, 869.7000, 881.7574;
step seven, detecting the metabolic molecule calibrator containing the internal standard again, and secondarily confirming the accurate molecular weight of the peak of the metabolic molecule calibrator under the negative ion detection mode except the known theoretical molecular weight by taking the pure product calibrator and the peak with the definite structure and the theoretical molecular weight as the internal standard, wherein the value is the average value of the tested 10-20 sites;
step eight, selecting peaks with high intensity and stability from peaks with known theoretical molecular weight or unknown theoretical molecular weight but with accurate molecular weight, establishing a molecular weight calibration list in mass spectrometer control software, wherein the molecular weight calibration list in a negative ion mode is 255.2330, 281.2486, 303.2330, 419.2568, 599.3202, 806.5458, 885.5499, 878.6033, 888.6240, 906.6346, 1042.6717, 1070.7030 and the calibratable range can calibrate the range of 150-; the molecular weight calibration in positive ion mode is listed as 184.1097, 478.2330, 496.3398, 524.3711, 734.5694, 760.5851, 782.5676, 798.5410, 806.5670, 832.5832, 848.5566, 881.7574, and can calibrate the range of 150-900 Da;
FIG. 2 is a peak-appearing diagram of a pure internal standard substance mixed with a metabolic molecule calibrator in a negative ion mode. The abscissa is mass-to-charge ratio (m/z), without unit; the ordinate is peak intensity, without units.
It should be noted that: the molecular weight calibrator in this example was extracted from a tissue sample. The molecular weight calibration lists in the two detection modes are only one type, and can be selected according to the detection requirements, wherein the molecular weight calibration lists in the negative ion mode can be 255.2330, 283.2643, 303.2330, 419.2568 and 599.3202, or 419.2568, 599.3202, 806.5458, 885.5499, 878.6033, 888.6240, 906.6346, 1042.6717 and 1070.7030, the molecular weight calibration lists in the positive ion mode can be 184.1097, 437.1032, 478.2330 and 502.2330, or 725.5410, 780.5514, 808.5560, 832.5832 and 881.7574, and the detection range is uniformly covered on the premise.
When extracting molecular weight calibrators from serum samples, the molecular calibration list includes: the mass-to-charge ratio m/z in the negative ion mode is 152.9699, 255.2330, 279.2330, 283.2643, 303.2330, 367.1551, 435.2482, 465.3030, 616.4784, 642.4990, 687.5554, 726.6026, 752.6139, 778.5289, 794.5272, 833.5268, 861.5499, 885.5499, 904.6189 and 922.6131, and the range of 100-1000Da can be calibrated; the mass/charge ratio m/z in the positive ion mode is 184.1097, 437.1032, 459.1522, 478.2330, 502.2330, 546.2744, 635.1439, 659.2695, 678.5991, 690.4992, 725.5410, 780.5514 and 832.5827, and can be calibrated in the range of 150-900 Da; where the biological samples are saliva and urine samples, the molecular weight calibration list includes: the mass/charge ratio m/z in the negative ion mode is 57.97152, 61.9812, 84.98303, 89.0226, 96.9641, 119.9455, 124.0074, 135.9187, 146.9659, 167.0217, 219.1761, 255.2328, 265.1451 and 311.1662, and the range of 0-350Da can be calibrated; the mass/charge ratio m/z in positive ion mode is 74.2500, 91.0540, 103.9910, 170.0970, 238.3760, and can be calibrated in the range of 0-300 Da.
Biological samples for the preparation of molecular weight calibrators encompass: human or mammalian serum, plasma, milk, bile, cerebrospinal fluid, saliva, urine, sweat, cells, tissues, cell lysates, tissue extracts, bacterial lysates, fungal lysates, etc., and thus the molecular weight calibration list obtained by the method for preparing a molecular weight calibration product of a metabolic molecule according to the present invention may also be other molecular weight combinations.
Example 7 evaluation of calibration Effect of molecular weight internal Standard calibration method for Metabolic molecule calibrator
Step one, calibrating the molecular weight of a serum biological sample to be detected:
1) collecting, extracting or enriching metabolic molecules in serum;
2) dissolving 1 part of metabolic molecule calibrator extracted from serum by using a dissolving solution to obtain a metabolic molecule calibrator solution;
3) transferring the metabolic sample collected, extracted or enriched in serum to a sample loading platform, and simultaneously spotting the metabolic calibrator solution on the sample loading platform, wherein the serum sample points to be tested are distributed around or adjacent to the metabolic molecule calibrator points;
4) starting a mass spectrometer to collect data, calibrating the spectrometer in two detection modes by using a metabolic molecule calibrator, and storing the method;
5) after the instrument calibration is completed, performing spectrogram acquisition on a serum sample to be detected, confirming a molecular weight calibration list capable of stabilizing peaks in the sample to be detected, and performing internal standard calibration on the sample to be detected of each hole;
step two, evaluating the calibration effect:
1) performing secondary identification on the peak with higher peak intensity and more stable peak in the spectrogram of the sample to be detected, and confirming that the peak and the corresponding peak in the molecular weight calibration list are from the same molecule;
2) comparing the mass-to-charge ratio of the calibrated peaks of the internal molecular weight standards at multiple sites with the accurate molecular weights in the molecular weight calibration list, and calculating the calibration accuracy (ppm) by the formula of | m/z(molecular weight after calibration)-m/z(accurate molecular weight)|/m/z(accurate molecular weight)×106And is used for judging the molecular weight calibration effect.
FIG. 3 is a peak spectrum of a metabolite calibrator extracted from serum in the negative ion mode. The abscissa is mass-to-charge ratio (m/z), without unit; the ordinate is peak intensity, without units.
FIG. 4 is a peak spectrum of a metabolite calibrator extracted from serum in positive ion mode. The abscissa is mass-to-charge ratio (m/z), without unit; the ordinate is peak intensity, without units.
FIG. 5 shows the calibrated accuracy of the reference peak of a representative well calibrated by the molecular weight internal standard, wherein the calibrated accuracy of the internal standard is <20ppm, and the calibrated accuracy of the molecular weight internal standard within the range of 400-1000Da is <10 ppm.
Example 8 evaluation of calibration Effect of molecular weight external Standard calibration method for Metabolic molecule calibrator extracted from tissue
Dissolving 1 part of metabolic molecule calibrator extracted and prepared from tissues by using a dissolving solution to obtain a metabolic molecule calibrator solution;
secondly, spotting the metabolic molecule calibrator on 9 detection sites of 3 multiplied by 3 on the surface of the nano mass spectrum chip, wherein the aperture of the nano mass spectrum chip is 3mm, the hole spacing is 4.5mm, the central position is an external standard calibration hole, and 8 sites around the chip are evaluation holes for molecular weight calibration, wherein the evaluation holes are 0.5-2 mu L/site;
starting a mass spectrometer to acquire data, firstly, carrying out molecular weight external standard calibration on the mass spectrometer by using the metabolic molecule calibrator in the external standard calibration hole, and carrying out spectrogram acquisition on an evaluation hole of the molecular weight calibration after calibration;
selecting a plurality of peaks which are uniformly distributed in the molecular weight detection range in the spectrogram obtained from the evaluation hole, adding other peaks which are not used for molecular calibration except for the calibration peak used for molecular weight calibration, and calculating the theoretically accurate molecular weight of the peak by means of secondary identification;
step five, comparing the mass-to-charge ratio of the molecular weight in the 8 calibration evaluation site holes with the accurate molecular weight, and calculating the relative error (ppm) with the formula of | m/z(molecular weight after calibration)-m/z(accurate molecular weight)|/m/z(theoretical molecular weight)×106And is used for judging the molecular weight calibration effect.
As an example, the peaks used for the evaluation of the effect of molecular weight calibration can be selected from the following list: the theoretical mass-to-charge ratios in the negative ion mode are m/z 255.2330, 281.2330, 303.2330, 419.2568, 599.3202, 806.5458, 878.6033, 888.6240 and 906.6346, and the mass-to-charge ratios in the positive ion mode are m/z 478.2330, 506.2642, 756.5514, 782.5676 and 798.5410.
FIG. 6 is a graph showing the peak appearance of the metabolic molecular calibrator derived from tissue in the negative ion mode. The abscissa is mass-to-charge ratio (m/z), without unit; the ordinate is peak intensity, without unit.
FIG. 7 is a graph showing the peak appearance of the tissue-derived metabolite calibrator in the positive ion mode. The abscissa is mass-to-charge ratio (m/z), without unit; the ordinate is peak intensity, without units.
FIG. 8 is a graph of the accuracy of external standard calibration using tissue-derived metabolic molecule calibrators as evaluation sites. The abscissa is mass-to-charge ratio (m/z), without unit; the ordinate is the relative error in ppm.
As shown in FIG. 8, the external standard calibration accuracy of the molecular weight calibrator kit on the metabolic molecular region is 3X 3 in 9 detection wells, and the average value of the relative error of the molecular weight is less than 50 ppm.
Example 9 evaluation of calibration Effect of molecular weight external Standard calibration method for Metabolic molecule calibrator extracted from serum
Dissolving 1 part of metabolic molecule calibrator prepared by extracting serum by using a dissolving solution to obtain a metabolic molecule calibrator solution;
secondly, spotting the metabolic molecule calibrator on 70 detection sites on the surface of a nano mass spectrum chip, wherein the pore diameter of the nano mass spectrum chip is 3mm, the pore spacing is 4.5mm, the overall size is 70 multiplied by 25mm, the central position pore of 70 detection sites is an external standard calibration pore, the peripheral 69 sites are evaluation pores for molecular weight calibration, and the concentration is 0.5-2 muL/site;
starting a mass spectrometer to acquire data, firstly, carrying out molecular weight external standard calibration on the mass spectrometer by using a metabolic molecular calibrator in an external standard calibration hole, and then, carrying out spectrogram acquisition on an evaluation hole of the molecular weight calibration;
step four, selecting a plurality of peaks which are uniformly distributed in the molecular weight detection range in the spectrogram obtained in the evaluation hole, comparing the mass-to-charge ratios of the molecular weights in the 69 calibration evaluation holes with the accurate molecular weight, and calculating the relative error (ppm) according to the formula of | m/z(molecular weight after calibration)-m/z(accurate molecular weight)|/m/z(theoretical molecular weight)×106And is used for judging the molecular weight calibration effect.
As an example, the peaks used for molecular weight calibration effect evaluation can be selected from the following list: the theoretical mass-to-charge ratios in the negative ion mode are m/z 255.2330, 283.2643, 303.2330, 435.2482, 465.3030, 616.4784, 642.4990, 687.5554, 726.6026, 861.5499 and 885.5499, and the mass-to-charge ratios in the positive ion mode are m/z 437.1032, 478.2330, 502.2330, 725.5410, 780.5514 and 808.5560.
FIG. 9 is a graph of the accuracy of external standard calibration using serum-derived metabolic molecule calibrators as evaluation sites. The abscissa is mass-to-charge ratio (m/z), without unit; the ordinate is the relative error in ppm. As shown in fig. 9, the relative error averages for the molecular weights of 69 wells were <150 ppm.
While the present invention has been described with reference to several exemplary embodiments, it is understood that the terms used are words of description and illustration, rather than words of limitation. As the present invention may be embodied in several forms without departing from the spirit or essential characteristics thereof, it should also be understood that the above-described embodiments are not limited by any of the details of the foregoing description, but rather should be construed broadly within its spirit and scope as defined in the appended claims, and therefore all changes and modifications that fall within the metes and bounds of the claims, or equivalence of such metes and bounds are therefore intended to be embraced by the appended claims.
Claims (7)
1. A molecular weight calibrator kit for metabolic spectrum detection is characterized by comprising a metabolic molecular calibrator, a dissolving solution, a molecular weight calibration list and a nano mass spectrum chip; wherein, the proportion of the metabolic molecule calibrator and the dissolving solution is as follows: 1 part of the metabolic molecule calibrator corresponds to 1-5 parts of the dissolving solution, and the metabolic molecule calibrator is extracted from a biological sample, wherein the biological sample comprises: mammalian serum, plasma, milk, bile, cerebrospinal fluid, saliva, urine, sweat, cells, tissue, cell lysate, tissue extract, bacterial lysate, or fungal lysate;
the molecular weight calibration list for biological samples as tissue samples includes: mass to charge ratio in negative ion modem/z= 255.2330, 281.2486, 303.2330, 419.2568, 599.3202, 806.5458, 885.5499, 878.6033, 888.6240, 906.6346, 1042.6717, 1070.7030; mass to charge ratio in positive ion modem/z = 184.1097、478.2330、496.3398、524.3711、734.5694、760.5851、782.5676、798.5410、806.5670、832.5832、848.5566、869.7000、923.7470、949.7621；
The molecular weight calibration list for biological samples as serum samples included: mass to charge ratio in negative ion modem/z= 152.9699, 255.2330, 279.2330, 283.2643, 303.2330, 367.1551, 435.2482, 465.3030, 616.4784, 642.4990, 687.5554, 726.6026, 752.6139, 778.5289, 794.5272, 833.5268, 861.5499, 885.5499, 904.6189, 922.6131, and 100-1000Da range can be calibrated; mass to charge ratio in positive ion modem/z= 184.1097, 437.1032, 459.1522, 478.2330, 502.2330, 546.2744, 635.1439, 659.2695, 678.5991, 690.4992, 725.5410, 780.5514, 808.5560, 832.5827, 150 and 900Da range can be calibrated;
the biological samples are molecular weights of saliva and urine samplesThe alignment list includes: mass to charge ratio in negative ion modem/z= 57.97152, 61.9812, 84.98303, 89.0226, 96.9641, 119.9455, 124.0074, 135.9187, 146.9659, 167.0217, 219.1761, 255.2328, 265.1451, 311.1662, calibratable 0-350Da range; mass to charge ratio in positive ion modem/z= 74.2500, 91.0540, 103.9910, 170.0970, 238.3760, which can calibrate the 0-300Da range.
2. The molecular weight calibrator kit for metabolic profiling detection according to claim 1, wherein the dissolution solution comprises: water, methanol, ethanol, acetonitrile, isopropanol, acetone and a mixed solvent obtained by mixing more than two solvents.
3. A method for preparing a molecular weight calibrator kit for metabolic profiling according to claim 1, comprising the steps of:
step one, preparing a metabolic molecule calibrator: extracting metabolic molecules in different biological samples by using an extraction reagent to obtain a calibrator containing the metabolic molecules;
step two, measuring the molecular weight of the metabolic molecules in the metabolic molecule calibrator:
1) mixing the dissolved solution and the metabolic molecule calibrator to obtain a metabolic molecule calibrator solution, and adding a pure calibrator serving as an internal standard into the metabolic molecule calibrator solution to prepare a metabolic molecule calibrator solution containing the internal standard;
2) spotting the metabolic molecule calibrator solution containing the internal standard on a sample loading platform, and naturally drying;
3) putting the sample carrying table into a mass spectrometer for detecting a positive ion mode and a negative ion mode, and determining the molecular weight of each mass spectrum peak in the metabolic molecule calibrator solution under the two detection modes by using an internal standard method;
4) selecting a mass spectrum peak with high peak intensity and stability to carry out secondary mass spectrum detection, obtaining a secondary mass spectrum, and carrying out structure identification on the secondary mass spectrum to obtain theoretically accurate molecular weight;
5) detecting the metabolic molecule calibrator containing the internal standard again, taking the pure calibrator and the peak with definite structure and theoretical accurate molecular weight as the internal standard, secondarily confirming and correcting the accurate molecular weight of the peak of the metabolic molecule calibrator except the known theoretical molecular weight in two detection modes, and obtaining an accurate molecular weight list;
6) selecting high-intensity and stable peaks in the accurate molecular weight list, and establishing a molecular weight calibration list in mass spectrometer control software;
step three, preparing the kit according to the following formula: the formula comprises the following components: a metabolic molecule calibrator, a dissolving solution and a molecular weight calibration list; the proportion of the metabolic molecule calibrator to the dissolving solution is as follows: 1 part of metabolic molecule calibrator corresponds to 1-5 parts of the dissolution solution.
4. A method of using the molecular weight calibrator kit for metabolic profiling according to claim 1, comprising an internal standard calibration method and an external standard calibration method; the internal standard calibration method is used for metabolic molecules which are overlapped with the metabolic molecule calibration product in a sample to be detected, and the external standard calibration method does not stipulate the components of the sample to be detected.
5. The method of using the molecular weight calibrator kit for metabolic profiling according to claim 4,
the internal standard calibration method comprises the following steps:
step one, calibrating the molecular weight of a biological sample to be detected:
1) obtaining metabolic molecules in a biological sample to be detected;
2) dissolving 1 part of metabolic molecule calibrator by using a dissolving solution to obtain a metabolic molecule calibrator solution;
3) transferring the metabolic molecules in the biological sample to be detected to a sample carrying table, and simultaneously spotting the metabolic molecule calibrator solution on the same sample carrying table;
4) starting a mass spectrometer to collect data, calibrating the spectrometer in two detection modes by using a metabolic molecule calibrator and a molecular weight calibration list, and storing the method;
5) after the instrument calibration is completed, spectrogram acquisition is carried out on a sample to be detected, a molecular weight calibration list capable of stably generating peaks in the sample to be detected is confirmed, peaks which cannot stably generate peaks in the sample to be detected in the molecular weight calibration list of the kit are removed, and internal standard calibration is carried out on the sample to be detected in each hole through the corrected molecular weight calibration list;
step two, evaluating the calibration effect:
1) performing secondary identification on the peak with higher intensity and more stable in the spectrogram of the sample to be detected, and confirming that the peak and the corresponding peak in the molecular weight calibration list are from the same molecule;
2) comparing the mass-to-charge ratios of the calibrated peaks of the internal molecular weight standards at the multiple sites with the accurate molecular weights in the molecular weight calibration list, calculating the calibration accuracy, and obtaining the formula of the calibrated molecular weightsm/zOf exact molecular weightm/zI/exact molecular weightm/z×106For evaluating the molecular weight calibration effect;
the use method of the internal standard calibration method in the data analysis software comprises the following steps:
step one, acquiring a metabolic spectrogram of a sample:
1) collecting, extracting or enriching metabolic molecules in a biological sample;
2) transferring the metabolic sample collected, extracted or enriched in the biological sample to a sample loading platform, and starting a mass spectrometer to collect data;
step two, realizing internal standard calibration in data analysis software:
transferring the metabolic spectrogram acquired by the mass spectrometer into data analysis software, calling a molecular weight calibration list, matching standard molecular weights in the molecular weight calibration list with detected molecular weights in a sample mass spectrogram, fitting a calibration curve according to a linear equation or a quadratic equation, and applying the calibration curve to each metabolic spectrogram to realize calibration of a full spectrogram;
step three, evaluating the calibration effect:
comparing the mass-to-charge ratio of the peaks of the molecules in the spectrograms of the multiple samples after the molecular weight internal standard calibration with the accurate molecular weight in the molecular weight calibration list, and calculating the relative error, wherein the formula is the molecular weight after the calibrationm/zOf precise molecular weightm/zI/exact molecular weightIsm/z×106And is used for judging the molecular weight calibration effect.
6. The method for using the molecular weight calibrator kit for metabolic profiling detection according to claim 4, wherein the software for external standard calibration comprises: instrument detection control software; the use method of the external standard calibration method comprises the following steps:
step one, calibrating the molecular weight of a biological sample to be tested:
1) collecting, extracting or enriching metabolic molecules in a biological sample;
2) dissolving 1 part of the metabolic molecule calibrator by using a dissolving solution to obtain a metabolic molecule calibrator solution;
3) transferring the metabolic sample collected, extracted or enriched in the biological sample to a sample carrying table, simultaneously applying the sample of the metabolic calibrator solution to the sample carrying table, and distributing the sample points to be tested around or adjacent to the metabolic molecule calibrator points;
4) starting a mass spectrometer to acquire data, performing external standard calibration on the molecular weight of the mass spectrometer in a negative ion mode and a positive ion mode by using a metabolic molecule calibrator and a molecular weight calibration list, and performing spectrogram acquisition on a sample to be detected in a sample hole to be detected in two detection modes after calibration;
step two, evaluating the calibration effect:
1) performing secondary identification on a peak with higher peak intensity and more stable peak in a spectrogram of a sample to be detected, wherein the secondary identification can confirm the peak of the molecular structure and calculate the theoretical molecular weight of the peak;
2) comparing the mass-to-charge ratio of the peaks after molecular weight calibration at multiple sites with the theoretically accurate molecular weight, and calculating relative error according to the formula of the calibrated molecular weightm/zOf theoretical molecular weightm/zI/theoretical molecular weightm/z×106And is used for judging the molecular weight calibration effect.
7. The method for using the molecular weight calibrator kit for metabolic profiling detection according to claim 5 or 6, wherein the mass spectrometer comprises: a time-of-flight ion source mass spectrometer, a SIMS ion source mass spectrometer, or an atmospheric ambient ion source mass spectrometer; the sample loading table of the mass spectrometer is a conductive substrate and comprises a stainless steel target plate or a nano-structure chip.
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