CN112198217B - Absolute quantitative mass spectrometry imaging method based on in-situ liquid extraction - Google Patents
Absolute quantitative mass spectrometry imaging method based on in-situ liquid extraction Download PDFInfo
- Publication number
- CN112198217B CN112198217B CN202011088174.XA CN202011088174A CN112198217B CN 112198217 B CN112198217 B CN 112198217B CN 202011088174 A CN202011088174 A CN 202011088174A CN 112198217 B CN112198217 B CN 112198217B
- Authority
- CN
- China
- Prior art keywords
- extraction
- sample
- time
- analyte
- internal standard
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
Landscapes
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Electrochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention discloses a baseAn absolute quantitative mass spectrometry imaging method for in situ liquid extraction, comprising the steps of: adopting a sampling technology based on dynamic in-situ liquid extraction to extract each pixel point on the surface of a sample one by one, adding an internal standard substance into extract liquor, staying each pixel point for a period of time, and collecting an extraction time curve of signals of an analyte and the internal standard substance along with the change of time in the period of time; t of each pixel pointpFitting the extraction time curve after s to obtain fitting parameters; calculating according to the formula of the invention to obtain the absolute contents M of the analytes of different pixel points; and obtaining the absolute content distribution of the analytes on the surface of the sample according to the absolute content of the analytes of each pixel point on the surface of the sample. The method can realize absolute quantification of single pixel points, and the actual extraction efficiency of any endogenous analyte can be obtained by fitting and calculating the time curve and the mass transfer model acquired by each pixel point.
Description
Technical Field
The invention belongs to the field of mass spectrum detection pretreatment, and particularly relates to a sample acquisition, data processing and absolute quantitative calculation method aiming at the problem of accurate quantification of a single pixel point of mass spectrum imaging.
Background
Mass Spectrometry Imaging (MSI) is a novel molecular-scale Imaging technique. The characteristic of multi-channel simultaneous detection meets the high-throughput non-target analysis of proteomics and metabonomics requirements in system biology. Meanwhile, the obtained spatial distribution information of various molecules also meets the requirement of molecular mechanism probes of biology in a tissue functional region and a cell layer on spatial resolution. For example, the study of the mechanism of neural signal transduction in brain science relies on the tracking of the distribution of the content of various signal transduction substances in the brain in different brain functional areas; the identification of cancer cell metastasis cannot be separated from metabolomic analysis on a single cell scale within tissues. The most important part for realizing mass spectrum imaging is an ionization or sampling technology with spatial resolution capability, and the currently commonly used technologies include matrix-assisted laser desorption ionization (MALDI), Secondary Ion Mass Spectrometry (SIMS), desorption electrospray ionization (DESI), nano-flow desorption electrospray ionization (nanoDESI), micro-liquid nodal sampling (LMJSS) and the like. Unlike the three previous examples, both nanoDESI and LMJSS are spatially resolved sampling techniques based on in situ liquid extraction (active liquid extraction techniques). The sampling method performs space resolution sampling by forming micro-liquid nodes with the diameter from micron to sub-millimeter with the surface of a sample through micro liquid, is simpler and more flexible than other devices of ionization sampling methods, and can be used together with different mass spectrum ion sources to realize online mass spectrum imaging.
However, mass spectrometry still presents significant problems in accurate characterization and quantification compared to other mass spectrometry techniques, particularly the absolute quantification of analytes on the surface of biological tissues. Because the matrix on the surface of the imaged sample is complex and changeable, signals obtained by various mass spectrometry ionization sampling technologies with spatial resolution capability at different positions on the surface of the sample are difficult to accurately generate a determined corresponding relation with the concentration content of a substance at the position, so that the spatial change of the signal value of molecules generated by mass spectrometry is not equal to the spatial change of the content of the molecules, thereby reducing the reliability of a mass spectrometry image and the significance of the provided biological information. Compared with other traditional mass spectrometry imaging methods, the mass spectrometry imaging method based on in-situ liquid extraction is quantitatively superior. Because the standard sample can be conveniently introduced into the system through the extraction solution, the signal of each pixel point can be corrected by using an internal standard correction method to correct the matrix difference and quantify the matrix difference. However, the labeling in the extraction liquid can only be used for correcting the content of the extracted analytes, and the change of the extraction efficiency at different pixel points cannot be corrected, so that the true absolute content in the tissues cannot be restored by the mass spectrometry quantitative method. Therefore, development of new methods is required.
Disclosure of Invention
The invention aims to solve the technical problems of overcoming the defects and shortcomings in the background technology and providing a sample acquisition, data processing and absolute quantitative calculation method aiming at the problem of single-pixel accurate quantification in-situ liquid extraction mass spectrometry imaging.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
an absolute quantitative mass spectrometry imaging method based on in-situ liquid extraction comprises the following steps:
(1) adopting a sampling technology based on dynamic in-situ liquid extraction to extract each pixel point on the surface of the sample one by one, adding an internal standard substance into extract liquor, and finally obtaining the concentration of the internal standard substance as CsThe flow rate of the extraction liquid is u, each pixel point stays for a period of time, and an extraction time curve of the signals of the analyte and the internal standard substance changing along with the time in the period of time is collected;
(2) t of each pixel pointpAnd (5) fitting the extraction time curve after s according to the following formula to obtain fitting parameters a and b or c and d:
y＝ce-d×t (2)
Wherein y is the analyte mass spectrum peak height at different time points divided by the mass spectrum peak height of the internal standard substance at the corresponding time point, t is time, unit s;
(3) calculating according to the following formula to obtain the absolute contents M of the analytes of different pixel points:
wherein Sa,iIs front tpS mass spectrum peak height at acquisition time point i on the analyte extraction time curve, Ss,iThe mass spectrum peak height of the internal standard substance at the corresponding time point I, delta t is the acquisition time interval of the mass spectrum signal, I is tpExtraction time curve after s fitted from t according to equation (1) or (2)ps is integrated to infinity and, for equation (1): i ═ a; for equation (2): i ═ c/d;
(4) and obtaining the absolute content distribution of the analytes on the surface of the sample according to the absolute content of the analytes of each pixel point on the surface of the sample.
Further, said tpBetween 10-20 s.
Further, the residence time at each extraction point is 15-50 s.
Furthermore, the sampling technology based on dynamic in-situ liquid extraction is that extraction liquid is continuously pumped into a sampling probe, and meanwhile, the extraction liquid is continuously pumped into a mass spectrum through the vacuum degree of a mass spectrum ion source nozzle, a liquid node is formed at the probe tip, and extraction is realized when the liquid node is contacted with the surface of a sample.
Furthermore, the sampling probe comprises an inner and outer sleeve of a capillary tube, a double-hole quartz tube, a goose-shaped tube or a folded tube, and the mass spectrometry ion source comprises an electrospray ionization source or an atmospheric pressure chemical ionization source.
Furthermore, the scanning of the sampling probe on the surface of the sample is realized by controlling the relative position of the probe and the solid sample through a three-axis platform program.
Further, the sampling probe is moved at a speed of 0-1000 μm/s between the pixel point and the spot.
Furthermore, the internal standard substance is a standard sample of which the mass spectrum signal intensity is similar to that of the analyte, but the mass spectrum peak position is not interfered by the analyte peak in the sample.
Further, the internal standard includes an isotopic standard of the analyte or an analog of the analyte that is not present in the sample.
Compared with the prior art, the invention has the beneficial effects that:
aiming at the problem that mass spectrum imaging of a solid sample is difficult to be absolutely quantitative in the prior art, the invention provides a novel sampling, scanning imaging and quantitative correction method based on an in-situ liquid extraction technology. Compared with the characteristic that each pixel point only has a single mass spectrum signal in the traditional mass spectrum imaging, the method provided by the invention acquires the sampling time curve of each pixel point by using a dynamic micro-liquid extraction technology, so that the acquired data has more time dimension information besides space information. The time curve equation which best accords with dynamic liquid flow extraction is obtained through examining different liquid-solid extraction mass transfer kinetic models, a complete mass transfer time curve from 0 to infinity can be predicted through mass spectrum data of limited time dimension and the derived equation, and the absolute content of the calculated analyte at the pixel point is obtained.
The method has the advantages that: 1. absolute quantification by adding a standard sample to a solid sample is avoided; and 2, the surface labeling of the sample cannot be absolutely quantified by a single pixel point because the labeling is difficult to be uniformly distributed in space and the distribution area of the standard sample is difficult to be controlled to be small. The method can realize absolute quantification of a single pixel point; 3. due to the different microenvironment and location within the sample between the standard sample and the actual endogenous analyte, conventional surface labeling of the sample does not allow accurate extraction efficiency of the endogenous analyte (e.g., intracellular metabolites). The method can obtain the actual extraction efficiency of any endogenous analyte by fitting and calculating the time curve and the mass transfer model acquired by each pixel point.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a curve of the extraction time of lipid in brain tissue at a single pixel point and a fitted curve of its mass transfer equation;
FIG. 2 is a signal-time curve of an internal standard substance over extraction time for a single pixel at different tissue functional region positions;
FIG. 3 is a signal-time plot of a standard sample over extraction time after tissue labeling;
FIG. 4 is a comparison of a quantitative image (A) of lipids in cerebellar tissue obtained by the method of the present invention and a conventional mass spectrometric image (B);
FIG. 5 is a comparison of the quantitative results obtained from lipid analytes in the same small functional tissue region using conventional solvent extraction-liquid chromatography-mass spectrometry (LC-MS) with the quantitative results obtained from conventional tissue-spiked imaging (Q-MSI) and the absolute quantitative results obtained from the imaging method of the present invention.
Detailed Description
In order to facilitate understanding of the invention, the invention will be described more fully and in detail with reference to the accompanying drawings and preferred embodiments, but the scope of the invention is not limited to the specific embodiments below.
Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.
The absolute quantitative mass spectrum imaging method for in-situ liquid extraction adopts a micro-liquid node probe sampling technology. And placing the tissue sample with the flat surface on a three-axis platform, and controlling the relative position of the single probe and the sample through the three-axis platform. The three-axis platform is programmed to brake so that the probe can scan and move or stay on the surface of the sample at a certain speed. In the scanning process, the micro liquid junction formed by the probe and the surface of the sample always keeps constant liquid junction coverage area and no air bubble is generated. The extraction liquid is pumped into the probe by a pump at a certain flow rate, then the negative pressure generated by the mass spectrum ion source is absorbed into the ion source for detection, and the stability of the liquid node is adjusted by the flow rate of the injection pump. To the extract was added an internal standard compound of known concentration.
When the sample imaging is carried out, the probe scans the sample point by point, each pixel point stays for 15-50s, the probe moves to the next point at a certain speed (0-1000 mu m/s) after the extraction of one pixel point is completed, and the operations are repeated. Each pixel will yield a 15-50s signal versus time curve for the analyte and the internal standard (hereinafter referred to as extraction time curve).
Fick's law and the second-order rate law both theoretically describe the rate of mass transfer of an analyte between two phases, so the present invention derives from two models that the time profile of the extraction of the analyte from the sample solid phase to the liquid phase through the continuous liquid flow after the diffusion layer is built is:
y＝ce-d×t(Fick's law)
Wherein y is the analyte mass spectral peak height at different time points divided by the mass spectral peak height of the internal standard at the corresponding time point; t is time(s); a and b or c and d are fitting parameters.
From this model, it can be seen that, as long as there are a small number of data points, the present invention can fit the data by the above formula to obtain parameters a, b or c and d, thereby obtaining the extracted content of the analyte when the extraction time is infinite, i.e., the absolute content of the analyte.
From our experimental results, it was found that the mass transfer of the analyte from the brain tissue actual sample to the microfluidic node obeys the above two equations after 10-20s of extraction time, while there is a ramp up before 10-20s, which may be related to the fact that no stable mass transfer diffusion layer has been established between the sample and the liquid. And the time (t) of this risep) Also varies with the nature of the sample, in relation to how fast a stable two-phase interface is established between sample and extract, and the time of establishment of the diffusion layer varies from sample to sample, generally tpBetween 10 and 20s, the exact value can be chosen by pre-experiment. In our earlier experiments, t for most biological tissue samplespNot more than 20 s. Therefore, when the extraction time exceeds tpAfter s, the parameters a and b or c and d can be obtained by fitting the above equation to the curve only by obtaining a time curve for a small period of time, thereby obtaining a fitting formula from tps to infinite integration results (I):
for the second order rate theory equation: i ═ a; for Fick's law equation: i ═ c/d
In our implementation, 15s (t) will be usedp15s) (before the stable diffusion layer is established), the signal value of the internal standard in the whole time curve and the fitting parameter are calculated according to the following formula, and the absolute content of the analyte of the pixel point can be obtained:
wherein M is in-unit pixelTotal amount of upper analyte (ng); sa,iFor the mass spectrum peak height at unit acquisition time point i on the time curve of the analyte in the first 15S, Ss,iThe mass spectrum peak height of the internal standard substance at the corresponding time point I, delta t is the interval(s) between the acquisition time point and the point of the mass spectrum signal, I is the integral value of a fitted curve after the time curve fitting after 15s (from 15s integral to infinity), u is the flow velocity (mu L/s) of the extraction solution, CsThe concentration of the internal standard in the extract was (ng/. mu.L).
The probe scans the surface of the sample point by point to obtain mass spectrum signals, and the absolute content of the analyte at each pixel point can be obtained through calculation, and the absolute content distribution on the surface of the sample can be shown through a heat map.
The sample can be any solid sample having a flat surface, such as a paper wrapper, a box, a plant leaf, a tissue slice, a sheet, a textile, a scroll, and the like. The small molecule substances that can be detected by the method of the present invention are arbitrary, and the following examples are merely illustrative of various lipids used in tissues.
The quantitative detection range (sampling depth) of the object to be detected in the solid sample is within a depth range from the outermost layer of the sample to 250-280 mu m away from the outermost layer of the sample, namely, the object to be detected within the depth range from the outermost layer of the sample to 250-280 mu m can be quantitatively detected, and the sampling depth is slightly different according to the different surface permeability of the micro-liquid node on different solid samples. If the thickness of the sample is less than 250 μm (such as packaging paper, packaging box, picture, tissue slice and leaf, etc.), the quantitative detection range is the whole sample, that is, the analyte in the whole thickness range of the sample can be quantitatively detected. For example, the quantitative detection range in examples 1 to 3 is the entire sample. Calculating the volume of the solid sample according to the quantitative detection range to obtain the volume content (mu g/mm) of the substance to be detected in the solid sample3)。
The three-axis platform is a stepping electric platform which can linearly move in the xyz three directions, and the probe or sample stage is fixed on the three-axis platform to control the probe or sample stage. The precision and the moving range of the three-axis stepping motor depend on the size of an imaging sample and the imaging spatial resolution. Three-axis stepper motor platforms are well known to those skilled in the art and are commercially available in the general stepper motor market.
The scanning speed of the sampling probe is 0-1000 mu m/s. Only in the speed range, the micro-liquid junction formed by the probe is stable, and the micro-liquid junction always keeps constant liquid junction coverage area and no bubble is generated.
The micro-liquid node probe can be an inner sleeve and an outer sleeve of a capillary tube, a double-hole quartz tube, a goose-shaped tube, a folded tube and the like. The mass spectrometry ion source can be an electrospray ionization source, an atmospheric pressure chemical ionization source and the like.
Example 1
And (3) acquiring extraction time curves of different lipids at different brain tissue positions by utilizing LMJSS and fitting mass transfer models of the curves. The method comprises the following specific steps:
(1) LMJSS device: the sampling probe is a coaxial capillary (the size of the outer capillary is 251 μm I.D./356 μm O.D., and the size of the inner capillary is 100 μm I.D./163 μm O.D.), the outer capillary is connected with an injection pump, and the inner capillary is connected with an inlet of a mass spectrometry ion source. The connecting capillary length between the inner capillary and the ion source is kept as small as possible. Here 20 cm.
(2) And (3) processing of tissues: the cerebellum tissue was cryosectioned (-20) at a thickness of 20 μm, and a room temperature glass plate was placed adjacent to the cryosection, and the section was heat-blotted and flat-fit tightly on the plate.
(3) The extraction probe performs extraction sampling on tissues: and (4) placing the glass plate adhered with the tissue on a triaxial platform and fixing the glass plate. The microfluidic node probe was subjected to single-point extraction in the substantia nigra and white matter regions of brain tissue, respectively, and the extract was a 6% ammonia-methanol/acetonitrile (1:1, V: V) solution with 1ppm PC21:0/22:6 and 10ppm GlcCer (d18:2/16: 0). The syringe pump pumps the extraction solution onto the probe at a rate of 5. mu.L/min, and the balance of the liquid node is achieved by slightly adjusting the syringe pump flow rate. Each point extraction time lasted 1 min.
(4) Acquisition of single-point mass spectrum signal-time curve: the mass spectrum is circularly switched under the full scan mode of positive ions (+4500V) and negative ions (-3000V), and the scanning range of the mass spectrum is 500-1000 Da; the ion accumulation time was 60ms and the overall cycle time (i.e., the acquisition point time interval Δ t) was 300 ms.
(5) Extraction time curve fitting of single points and absolute quantification of analytes: and (3) plotting the ratio of the mass spectrum peak height of the collected analyte to the internal standard mass spectrum peak height at the corresponding time point for time to obtain a curve (simply called a time curve) of the relative mass spectrum peak height of the analyte along with the time. The time curves of different analytes at different positions are fitted according to the following formula 1, and the fitting time ranges are 15-22s, 15-25s, 15-33s and 15-60 s. The fitting parameters obtained by fitting are substituted into formula 2 to calculate the content of the analyte in the single pixel point.
(6) And (4) analyzing results: the extraction time curve of analyte m/z 760.5 at a single pixel point and its fit curve for different time periods 15s later are shown in figure 1. Fitting degree R through fitting of different time periods2And the absolute contents of the analytes on the single points calculated according to the fitting parameters are respectively displayed on the fitting curves. As can be seen from the figure, the derived mass transfer model can be well matched with the actually measured analyte extraction time curve, and the fitting degree is higher than 0.9. In addition, comparison of the analyte levels obtained from the shorter time curve fit with the all time curve fit after 15s (15-60s) shows that even using time curve data from 15-25s gives results very close to the quantitative results calculated for the 15-60s time curve (error less than 10%). Therefore, the absolute content of the analyte can be predicted through the model only by the extraction time of the single pixel point of 25 s. The signal changes over the extraction time for the internal standard (m/z 876.6488 and m/z 736.5358) and the analyte (m/z 760.5808) are shown in FIG. 2. As can be seen, although the concentration of the internal standard in the extract at any time point is the same, it is not excludedThe signal of (a) fluctuates with time, the largest fluctuation is at the peak of the analyte signal, and it can be seen from the figure that the signal of the internal standard substance shows a low valley when the signal of the analyte reaches the peak, and the signal of the internal standard substance slowly rises back to the level corresponding to the signal at the time point 0 as the signal of the analyte decreases. This is mainly due to the fact that the matrix effect of the extraction liquid is high after a large amount of analyte is extracted from the tissue, and thus the signals of all substances, including the internal standard substance in the extraction liquid, are severely suppressed, so that the signals of the internal standard substance reflect the matrix effect in the extraction liquid in real time. Therefore, the relative signal obtained by dividing the analyte signal by the internal standard signal has good correction to the matrix effect of the extraction liquid, and can reflect the real concentration of the analyte in the extraction liquid.
Example 2
The extraction time curve of the lipid standard sample dripped on the brain tissue is collected by using LMJSS. The method comprises the following specific steps:
(1) LMJSS device: in accordance with example 1.
(2) And (3) processing of tissues: the cerebellum tissue was cryosectioned (-20) to a thickness of about 20 μm, and a room temperature glass plate was placed adjacent to the cryosection, and the section was heat-blotted and held flat and tightly against the plate. Internal standards 1ppm PC21:0/22:6 and 10ppm GlcCer (d18:2/16:0) were applied to the tissue surface.
(3) The extraction probe performs extraction sampling on tissues: and placing the glass plate adhered with the tissue on a triaxial platform and fixing the glass plate. The micro liquid node probes respectively perform single-point extraction in the tissue area on which the internal standard substance is dripped, and the extract liquor is 6% ammonia water-methanol/acetonitrile (1:1, V: V) solution. The syringe pump pumps the extraction solution onto the probe at a rate of 5. mu.L/min, and the equilibrium of the liquid node is achieved by slightly adjusting the syringe pump flow rate. Each point extraction time lasted 2.5 min.
(4) Acquisition of single-point mass spectrum signal-time curve: in accordance with example 1.
(5) And (4) analyzing results: the pair of time curves for the internal standard and the analytes PC36:1 and PC32:0 dropped on the tissue were extracted is shown in fig. 3. As can be seen from the figure, the difference between the extraction time curve of the internal standard substance and the extraction time curve of the analyte under the same conditions is large, the internal standard substance is completely extracted into the solution within 0.1min, and the analyte needs 1min for extraction until 90% is extracted. Therefore, if the traditional manner of dripping the internal standard substance on the surface of the tissue is adopted, and the quantification is carried out through the ratio of the signals of the analyte and the internal standard substance at a single time point, the real content ratio of the analyte and the internal standard substance on the tissue cannot be obtained at all, and the real content of the analyte cannot be obtained. Thereby illustrating the advantages of the method of the present invention.
Example 3
Absolute quantitative mass spectrometry imaging of lipids in cerebellar tissue is performed using a single probe and a novel quantitative calibration method. The method comprises the following specific steps:
(1) single probe device: the sampling probe is a double-hole glass tube elongated by heat. The glass tube was elongated to have a tip diameter of 100 μm, and quartz capillaries having dimensions of 251 μm I.D./356 μm O.D. and 100 μm I.D./163 μm O.D. were inserted into two holes at the other end, respectively. The capillary tube and the glass tube are sealed and fixed by ultraviolet curing glue. A251 μm inner diameter capillary was connected to a syringe pump and a 100 μm inner diameter capillary was connected to the mass spectrometer ion source inlet. The connecting capillary length between the capillary holding the 100 μm internal diameter and the ion source is kept as small as possible. Here 20 cm.
(2) And (3) processing of tissues: in accordance with example 1.
(3) The extraction probe performs extraction sampling on tissues: and placing the glass plate adhered with the tissue on a triaxial platform and fixing the glass plate. A single probe was scanned point-by-point over a rectangular block of cerebellar tissue, and the extracts were 6% ammonia-methanol/acetonitrile (1:1, V: V) solutions with 1ppm PC21:0/22:6 and 10ppm GlcCer (d18:2/16: 0). The syringe pump pumps the extraction solution onto the probe at a rate of 5. mu.L/min, and the balance of the liquid node is achieved by slightly adjusting the syringe pump flow rate. Each point extraction time lasted 30 s.
(4) Acquisition of single-point mass spectrum signal-time curve: in accordance with example 1.
(5) Extraction time curve fitting of single points and absolute quantification of analytes: the time curves for different analytes at different locations were fitted according to equation 1 below, with time curves ranging from 15-30s for each point. The fitting parameters obtained by fitting are brought into formula 2 to calculate the content of the analyte in the single pixel point.
(6) Absolute quantification of lipids in functional regions of tissues using traditional LC-MS methods: the tissue of the cerebellum in the region shown in FIG. 4(A) was dug out, and frozen and sliced, the obtained slice was extracted with methanol/chloroform (1:2) solvent, and the extract was injected into LC-MS for analysis. The assay results were quantified using standard addition methods.
(7) And (4) analyzing results: the comparison of the imaging graph of various lipids of the cerebellar tissue detected by the novel quantitative mass spectrometry imaging method with the imaging graph drawn by the conventional mass spectrometry signal is shown in fig. 4. As can be seen from fig. 4, the distribution of different lipids between the substantia nigra and the white matter of the cerebellum is very different, and the difference is shown in the quantitative mass spectrometry (fig. 4A) and the conventional mass spectrometry (fig. 4B), for example, the glycosphingolipid (m/z 848.64) is about 5 times higher in the root (lower right corner region) than in the branch (upper left corner region) of the cerebellum fiber layer in the quantitative mass spectrometry, while the signals of both regions are weaker and the difference is not so large in the normal mass spectrometry. And the difference of the distribution of glycosphingolipids in white matter and substantia nigra of the cerebellum can be clearly seen in the quantitative mass spectrum imaging picture, but the normal imaging picture is fuzzy. These are mainly due to mass transfer kinetics differences of lipids at different locations that are not corrected for in normal mass spectrometry imaging. The quantitative results of the conventional LC-MS method are compared with those obtained by imaging the same region using a new quantitative mass spectrometer, as shown in fig. 5. As can be seen from FIG. 5, the quantification results of the conventional lipid quantification method are substantially consistent with those of the quantitative mass spectrometry imaging method, whereas the novel method of the present invention has a spatial resolution of 100 μm.
The foregoing is considered as illustrative of the preferred embodiments of the invention and is not to be construed as limiting the invention in any way. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical spirit of the present invention should fall within the protection scope of the technical scheme of the present invention, unless the technical spirit of the present invention departs from the content of the technical scheme of the present invention.
Claims (8)
1. An absolute quantitative mass spectrometry imaging method based on in-situ liquid extraction is characterized by comprising the following steps:
(1) adopting a sampling technology based on dynamic in-situ liquid extraction to extract each pixel point on the surface of the sample one by one, adding an internal standard substance into the extract liquor, wherein the concentration of the internal standard substance is CsThe flow rate of the extraction liquid is u, each pixel point stays for a period of time, and an extraction time curve of the signals of the analyte and the internal standard substance changing along with the time in the period of time is collected;
(2) t of each pixel pointpThe extraction time curve after s is fitted as follows, said tps is between 10 and 20s, and fitting parameters a and b or c and d are obtained:
y＝ce-d×t (2)
Wherein y is the analyte mass spectrum peak height at different time points divided by the mass spectrum peak height of the internal standard substance at the corresponding time point, and t is time and unit s;
(3) calculating according to the following formula to obtain the absolute contents M of the analytes of different pixel points:
wherein Sa,iIs front tpMass spectrum peak at collection time point i on the analyte extraction time curve in sHigh, Ss,iThe mass spectrum peak height of the internal standard substance at the corresponding time point I, delta t is the acquisition time interval of the mass spectrum signal, I is tpThe extraction time curve after s is fitted according to the formula of step (2) to obtain a fitted curve from tps is integrated to infinity and, for equation (1): i ═ a; for equation (2): i ═ c/d;
(4) and obtaining the absolute content distribution of the analytes on the surface of the sample according to the absolute content of the analytes of each pixel point on the surface of the sample.
2. The method of in situ liquid extraction based absolute quantitative mass spectrometry imaging according to claim 1, wherein each extraction point residence time is 15-50 s.
3. The absolute quantitative mass spectrometry imaging method based on in-situ liquid extraction as claimed in any one of claims 1 to 2, wherein the dynamic in-situ liquid extraction based sampling technique is to pump the extraction liquid into the sampling probe continuously, and simultaneously pump the extraction liquid into the mass spectrum continuously by the vacuum degree of the mass spectrometry ion source nozzle, so that a liquid node is formed at the probe tip, and the extraction is realized when the liquid node is in contact with the surface of the sample.
4. The method of claim 3, wherein the sampling probe comprises an inner and outer capillary tube, a double-hole quartz tube, a goose-shaped tube or a folded tube, and the mass spectrometry ion source comprises an electrospray ionization source or an atmospheric pressure chemical ionization source.
5. The method of claim 3, wherein the sampling probe is scanned on the surface of the sample by controlling the relative position of the probe and the solid sample by a three-axis platform program.
6. The method of claim 3, wherein the sampling probe is moved between the pixel point and the spot at a speed of 0-1000 μm/s.
7. The in-situ liquid extraction-based absolute quantitative mass spectrometry imaging method according to any one of claims 1-2, wherein the internal standard substance is a standard sample with a mass spectrum signal intensity similar to that of the analyte, but a mass spectrum peak position not interfered by the analyte peak in the sample.
8. The method of in situ liquid extraction based absolute quantitative mass spectrometry imaging of claim 7, wherein the internal standard comprises an isotopic standard of the analyte or an analog of the analyte that is not present in the sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011088174.XA CN112198217B (en) | 2020-10-13 | 2020-10-13 | Absolute quantitative mass spectrometry imaging method based on in-situ liquid extraction |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011088174.XA CN112198217B (en) | 2020-10-13 | 2020-10-13 | Absolute quantitative mass spectrometry imaging method based on in-situ liquid extraction |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112198217A CN112198217A (en) | 2021-01-08 |
| CN112198217B true CN112198217B (en) | 2022-05-20 |
Family
ID=74008564
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011088174.XA Active CN112198217B (en) | 2020-10-13 | 2020-10-13 | Absolute quantitative mass spectrometry imaging method based on in-situ liquid extraction |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112198217B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116429870A (en) * | 2022-09-30 | 2023-07-14 | 上海立迪生物技术股份有限公司 | Method for eliminating imaging mass spectrum flow type sensitivity difference |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106442701A (en) * | 2016-11-28 | 2017-02-22 | 山东省分析测试中心 | Mass spectrometry method based on single-pixel solid-phase extraction technology and application thereof |
| CN206192944U (en) * | 2016-11-28 | 2017-05-24 | 山东省分析测试中心 | A single pixel solid -phase extraction array panel for sample pretreatment in mass spectrum formation of image and formation of image analytic system thereof |
| CN110568056A (en) * | 2019-09-05 | 2019-12-13 | 中南大学 | In-situ mass spectrometry quantification method for small molecular substances on solid surface |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8796654B2 (en) * | 2008-07-31 | 2014-08-05 | Shimadzu Coporation | Scan device for microscope measurement instrument |
| US20110124398A1 (en) * | 2008-11-11 | 2011-05-26 | Templeman Michael S | Card and dice wagering game |
-
2020
- 2020-10-13 CN CN202011088174.XA patent/CN112198217B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106442701A (en) * | 2016-11-28 | 2017-02-22 | 山东省分析测试中心 | Mass spectrometry method based on single-pixel solid-phase extraction technology and application thereof |
| CN206192944U (en) * | 2016-11-28 | 2017-05-24 | 山东省分析测试中心 | A single pixel solid -phase extraction array panel for sample pretreatment in mass spectrum formation of image and formation of image analytic system thereof |
| CN110568056A (en) * | 2019-09-05 | 2019-12-13 | 中南大学 | In-situ mass spectrometry quantification method for small molecular substances on solid surface |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112198217A (en) | 2021-01-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110568056B (en) | In-situ mass spectrometry quantification method for small molecular substances on solid surface | |
| JP4381820B2 (en) | Micro extractor for biological tissue examination | |
| US8829430B2 (en) | Pulsed mass calibration in time-of-flight mass spectrometry | |
| Leinonen et al. | Liquid-phase microextraction for sample preparation in analysis of unconjugated anabolic steroids in urine | |
| US20120053065A1 (en) | Device for high spatial resolution chemical analysis of a sample and method of high spatial resolution chemical analysis | |
| Deng et al. | Slug-flow microextraction coupled with paper spray mass spectrometry for rapid analysis of complex samples | |
| CN104134606A (en) | Microextraction probe electrospray ion source and manufacturing method and application of microextraction probe electrospray ion source | |
| CN112198217B (en) | Absolute quantitative mass spectrometry imaging method based on in-situ liquid extraction | |
| CN109406687B (en) | Method for detecting double phospholipids at high flux | |
| CN108872415A (en) | The analyzing detecting method of monohydroxy polycyclic aromatic hydrocarbon in a kind of urine | |
| Rosting et al. | High-throughput analysis of drugs in biological fluids by desorption electrospray ionization mass spectrometry coupled with thin liquid membrane extraction | |
| Cabal et al. | Determination of ring-substituted amphetamines through automated online hollow fiber liquid-phase microextraction-liquid chromatography | |
| Catai et al. | Efficient and highly reproducible capillary electrophoresis–mass spectrometry of peptides using Polybrene‐poly (vinyl sulfonate)‐coated capillaries | |
| Neu et al. | Development and characterization of a capillary gap sampler as new microfluidic device for fast and direct analysis of low sample amounts by ESI-MS | |
| Luo et al. | Per-pixel absolute quantitation for mass spectrometry imaging of endogenous lipidomes by model prediction of mass transfer kinetics in single-probe-based ambient liquid extraction | |
| CN113030361A (en) | Metabonomics analysis method for treating aqueous-phase PPCPs by using solid-phase extraction column based on UHPLC-MS/MS technology | |
| CN113433205A (en) | Imaging quality analysis system and analysis method using imaging quality analysis | |
| Busch | Mass spectrometric detection for thin-layer chromatographic separations | |
| Dvořák et al. | In‐line coupling of supported liquid membrane extraction across nanofibrous membrane to capillary electrophoresis for analysis of basic drugs from undiluted body fluids | |
| CN109932354A (en) | It is a kind of for the separation and concentration device in situ of Surface enhanced Raman spectroscopy trace analysis and application | |
| Schappler et al. | 18 Coupling CE and microchip-based devices with mass spectrometry | |
| CN112540139B (en) | Molecular weight calibrator kit for metabolic profiling detection, and preparation method and use method thereof | |
| CN114527154A (en) | Method for metabolomics sample component detection | |
| CN116148367A (en) | Method for detecting 23 pesticide metabolites in urine based on liquid-liquid extraction | |
| Midey et al. | Spatial mapping of cellular metabolites using DESI ion mobility mass spectrometry |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |