JP4519675B2 - Liquid chromatograph mass spectrometer and liquid chromatograph mass spectrometer - Google Patents

Description

  The present invention relates to a liquid chromatograph mass spectrometry method for analyzing acidic sugar chains such as glycoproteins and neutral sugar chains.

  The acidic sugar chains (sialylated sugar chains) and neutral sugar chains of glycoproteins present in biological components have recently attracted attention in connection with many diseases.

  As a method for simultaneous analysis of these sugar chains, the sugar chain of the glycoprotein is released, the reducing end of the sugar chain is derivatized with a reagent such as 2-aminopyridine, and then liquid chromatography using multiple columns with different elution principles. There is a method of analyzing by a graph (HPLC).

  In this method, sugar chains are identified by plotting the elution times of sugar chains from a plurality of columns and comparing them with the elution times of known sugar chains.

  In addition, in recent years, a sugar chain analysis method using a liquid chromatograph mass spectrometer (LC / MS) using a mass spectrometer as a detector for liquid chromatography has also been performed (Journal of American Society Mass Spectrometry, 13: 1322-1330 (2002)).

  A mass spectrometer is not only highly sensitive, but can also acquire structural information at the same time, so it is suitable for analysis of sugar chains having complex and similar structures.

  In the analysis by LC / MS, LC / ESI-MS using an electrospray ion source (ESI) for ionization is widely used.

  Here, as described in Patent Document 1, since a high voltage is applied between the capillary and the sampling orifice, ESI requires an electric shock prevention measure, and the apparatus structure becomes complicated.

  Therefore, a sonic spray ion source is described in which ionization is performed by spraying an eluent at a high speed, rather than applying high voltage to the capillary tip.

JP-A-8-62200 Analytical Biochemistry, 171, 73-90 (1988)

  By the way, in order to shorten the analysis time, it is required to simultaneously analyze an acidic sugar chain and a neutral sugar chain.

  However, for simultaneous analysis of acidic sugar chains and neutral sugar chains by LC / ESI-MS, there is a large difference in ionization efficiency between acidic sugar chains and neutral sugar chains by ESI. The components cannot be analyzed at the same time with high sensitivity.

  As a means for solving this problem, there is a method of equalizing the ionization efficiency of the neutral sugar chain and the acidic sugar chain by derivatizing the acidic sugar chain by a complete methylation method or a methyl esterification method.

  However, this method has drawbacks such that a sample amount is required for derivatization and that structural information is difficult to obtain because enzyme digestion with sialidase becomes impossible.

  In addition, there is a method in which the neutral sugar chain is analyzed by LC / ESI-MS in the positive ion measurement mode and the acidic sugar chain is analyzed in the negative ion measurement mode without derivatization as described above.

  In this case, structural information is easily obtained because derivatization of the sugar chain is not performed, but it becomes necessary to analyze the neutral sugar chain and the acidic sugar chain after separating them with an ion exchange column, etc., so the analysis time can be shortened. It cannot be planned.

  In addition, a method of simultaneously analyzing a neutral sugar chain and an acidic sugar chain in a positive ion measurement mode using a strongly acidic eluent condition (for example, 0.15% formic acid solution, pH of about 2.5) is also performed. Under strongly acidic conditions, both neutral sugar chains and acidic sugar chains suppress the dissociation of hydrogen ions, and hydrogen-added ions are likely to be generated when analyzing in the positive ion measurement mode.

  However, it is known that ionization efficiency in ESI generally decreases under strongly acidic conditions, and it is difficult to realize high sensitivity analysis. In addition, sialic acid, which is a partial structure of acidic sugar chains, may be released under strongly acidic conditions, which is not practical as analysis conditions for acidic sugar chains.

  Further, the method using the sonic spray ion source described above does not consider the simultaneous analysis of neutral sugar chains and acidic sugar chains with high sensitivity. In the prior art, neutral sugar chains and acidic sugar chains are not considered. It was difficult to realize a technique for simultaneously analyzing

  An object of the present invention is to realize a liquid chromatograph mass spectrometric method that simultaneously analyzes a neutral sugar chain and an acidic sugar chain with high sensitivity by LC / MS.

  In order to achieve the above object, the present invention uses a sonic spray ion source (SSI) in the ionization method of LC / MS, and the signal intensity obtained for both neutral sugar chains and acidic sugar chains is high, and both signal intensities are obtained. We were able to find a condition where the difference between them was small.

  As a result, a method of simultaneously analyzing neutral sugar chains and acidic sugar chains with high sensitivity by LC / MS could be realized.

  That is, the present invention is configured as follows.

(1) In a liquid chromatograph mass spectrometry method, a sample containing a neutral sugar chain and an acidic sugar chain is sent to a separation column with an eluent having a pH of 3 to 5, and the sample is separated into each component by the separation column. the samples were separated, separated into each component by spraying in substantially sonic speed, and feeding the sonic spray ion source for ionizing the ion source is operated in the negative ion measurement mode, in the said sample The sex difference sugar chain and the acidic sugar chain are ionized, and the ionized sample is subjected to mass spectrometry.

(2) In a liquid chromatograph mass spectrometer, a plurality of eluents having different pHs are mixed so as to have a pH of 3 to 5, and a liquid feed pump for feeding the liquid, and an eluent fed by the liquid feed pump to a sample introduction means for introducing a sample containing a neutral sugar chain and an acidic sugar chain, and separation column for separating said sample sent by the eluant to each component, the sample separated by the separation column, substantially A sonic spray ion source that ionizes neutral differential sugar chains and acidic sugar chains in the sample by spraying at a speed of sound , and a mass spectrometric unit that performs mass analysis on the sample ionized by the ion source.

(3) Preferably, in (1) and (2) above, the separation column is a reverse phase column.
(4) Preferably, in the above (1) and (2), the separation column is a silica column, and the eluent is a mixed solution of an ammonium acetate aqueous solution and an acetonitrile aqueous solution. The sample is fed to the separation column while changing the mixing ratio of the ammonium acetate aqueous solution and the acetonitrile aqueous solution.

  A liquid chromatograph mass spectrometric method and apparatus for simultaneous analysis of neutral sugar chains and acidic sugar chains with high sensitivity by LC / MS can be realized.

  Since simultaneous analysis of neutral sugar chains and acidic sugar chains is possible, the analysis efficiency is improved. Moreover, sample consumption can be reduced.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings.
FIG. 1 is a schematic configuration diagram of a liquid chromatograph mass spectrometer (LC / MS) to which an embodiment according to the present invention is applied.

  In FIG. 1, two types of eluents 2 and 3 are fed by the liquid feed pump 1 while changing the mixing ratio at the start of measurement. A sample solution containing neutral sugar chains and acidic sugar chains is introduced by the sample introduction means 4 and sent to the separation column 5 by the eluent.

  The sugar chain components in the sample solution are separated for each component by an interaction such as adsorption / distribution in the separation column 5 and sent to a sonic spray ion source 6 which is an ionization means.

  In the sonic spray ion source 6, the glycan component is ionized by spraying an eluent containing the glycan component with a gas flow (sonic spray) at a substantially sonic velocity.

  A voltage (about 0 to 1 kV) can be applied to the sonic spray ion source 6 by a high voltage power source 7 which is a driving power source. The mass number and the signal intensity of the sugar chain component ionized by the sonic spray ion source 6 are detected by the mass spectrometer 8.

  Here, when a reverse phase column such as a C18 silica column or a C30 silica column is used as the analysis column 5, it is often used while mixing an organic solvent such as water and acetonitrile as an eluent. The pH of the eluents 2 and 3 greatly affects the signal intensity at the apparatus 8.

  When the glycan component is measured in the negative ion measurement mode using the sonic spray ion source 6, since the acidic glycan contains sialic acid in its structure, the eluent is easily a negative ion under neutral conditions. Varicella desorption ions are generated and can be detected with high sensitivity by the mass spectrometer 8.

  On the other hand, when the eluent becomes acidic, the hydrogen ion concentration in the eluent is high, so that hydrogen desorbed ions are less likely to be generated, and accordingly, high-sensitivity detection with the mass spectrometer 8 becomes difficult.

  Therefore, the inventor of the present application examined the pH in the eluent and the signal intensities of neutral sugar chains and acidic sugar chains.

  As a result, the separation column 5 is equipped with a C30 silica column, the eluent 2 is a 1 mmo1 / L ammonium acetate aqueous solution, the eluent 3 is a 10% acetonitrile aqueous solution containing 1 mmo1 / L ammonium acetate, and the pH is adjusted under slightly acidic conditions (for example, pH 4 It was found that by using the one adjusted in the vicinity of .3), a high-sensitivity analysis condition can be obtained at the same time without impairing the separation of neutral sugar chains and acidic sugar chains in the negative ion measurement mode and an applied voltage of 0 kV.

  FIG. 2 is a graph showing an example of the results of the examination experiment conducted in finding the above-described measurement conditions, where the vertical axis shows the signal intensity and the horizontal axis shows the measurement mode and pH.

  In FIG. 2, 200.4 is the name of one component of the neutral sugar chain, and 2A1-200.4 is the name of one component of the acidic sugar chain (FIG. 3 shows the structural formula of each sugar chain component). ).

  The bar graph on the left side of FIG. 2 (3 data for each of the neutral sugar chain and the acidic sugar chain) shows the neutral sugar chain when the liquid chromatograph mass spectrometer shown in FIG. 1 is used in the positive ion measurement mode. The signal intensity of each component of the acidic sugar chain is shown.

  The bar graph on the right side of FIG. 2 (three data for each of neutral sugar chains and acidic sugar chains) shows the neutral sugar chains when the liquid chromatograph mass spectrometer shown in FIG. 1 is used in the negative ion measurement mode. And the signal intensity of each component of the acidic sugar chain.

  In addition, FIG. 2 shows the result of measuring sugar chain components by changing the eluent conditions to weakly acidic (pH 4.3), neutral (pH 6.5), and basic (pH 8.6) for each measurement mode. It is.

  As a result of the positive ion measurement mode, the signal intensity of the neutral sugar chain (200.4) was the highest among all conditions when the eluent condition was basic (pH 8.6).

  However, the difference in signal intensity between the neutral sugar chain (200.4) and the acidic sugar chain (2A1-200.4) is large under any eluent conditions. In addition, since the signal strength of acidic sugar chains is small, it cannot be said that it is suitable for simultaneous analysis of neutral sugar chains and acidic sugar chains.

  In contrast, in the result of the negative ion measurement mode, the difference in signal intensity between the neutral sugar chain and the acidic sugar chain is small regardless of the eluent conditions. In addition, by making the eluent weakly acidic (pH 4.3), both the neutral sugar chain and the acidic anchor chain were detected with high signal intensity and high sensitivity.

  Therefore, in one embodiment of the present invention, mass spectrometry is performed with the neutral sugar chain and the acidic sugar chain in the negative ion measurement mode and the pH of the eluent is weakly acidic (pH 4.3).

  Using one embodiment of the present invention, neutral sugar chains and acidic sugar chains obtained by releasing a sugar chain of a glycoprotein in serum and derivatizing the reducing end of the sugar chain with a 2-aminopyridine reagent The results of measuring the mixed solution are shown in FIG.

  4 (a) is a total ion chromatogram obtained by a mass spectrometer, and FIG. 4 (b) is a mass chromatogram obtained by extracting ions having mass numbers corresponding to neutral sugar chains and acidic sugar chains. It is.

  During the retention time of 22 to 42 minutes, it is possible to detect a total of 8 sugar chains including 3 neutral sugar chains (2 components in m / z 850) and 5 acidic sugar chains in a single measurement. It was.

  Thus, according to the present invention, it has become possible to realize high-sensitivity simultaneous analysis of neutral sugar chains and acidic sugar chains, which was difficult with the prior art.

  According to the results of the study by the present inventors, in the negative ion measurement mode, the signal strength between the neutral sugar chain and the acidic sugar chain is high in the pH range of 3 to 5, and the difference between the two signal strengths is different from each other. It is small and can be analyzed simultaneously with high sensitivity.

  As another embodiment of the present invention, there is a liquid chromatograph mass spectrometer capable of adjusting the pH of an eluent according to a measurement target.

  As shown in FIG. 1, the pump 1 can change the mixing ratio of the eluents 2 and 3. Therefore, if the pH of the eluents 2 and 3 is set in advance, the pH of the eluent can be adjusted by setting the mixing ratio to an appropriate value.

  Specifically, when the pH of the eluent is set by a setting means (keyboard or the like) and a controller (not shown), the liquid feed pump 1 supplies the eluent at a predetermined mixing ratio so as to reach the pH. Mix.

  Alternatively, the pH of the mixed eluent is measured by a pH measurement sensor, and the liquid feed pump 1 can adjust the mixing ratio of the eluent so as to obtain a desired pH.

  According to the liquid chromatograph mass spectrometer according to the present invention, the pH of the eluent can be adjusted to the optimum range according to the analysis target of the sample, so that neutral sugar chains and acidic sugar chains can be increased by LC / MS. In addition to being able to perform simultaneous analysis with sensitivity, even when only neutral sugar chains or only acidic sugar chains are targeted, the eluent is optimally analyzed according to the target and analyzed with high sensitivity. It becomes possible.

It is a schematic block diagram of the liquid chromatograph mass spectrometer (LC / MS) to which this invention is applied. It is a graph which shows the result of having changed pH in the positive / negative ion measurement mode using the sonic spray ion source about the sugar_chain | carbohydrate component. It is a figure which shows the structural formula of the neutral sugar chain from which the measurement result shown in FIG. 2 was obtained, and an acidic sugar chain. It is a figure which shows the simultaneous analysis result of the neutral sugar chain obtained by processing serum using one Embodiment of this invention, and an acidic sugar chain.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Liquid feed pump 2, 3 Eluent 4 Sample introduction means 5 Analysis column 6 Sonic spray ion source (SSI)
7 High-voltage power supply 8 Mass spectrometer

Claims (6)

A sample containing neutral sugar chains and acidic sugar chains is sent to a separation column with an eluent having a pH of 3 to 5,
The sample is separated into each component by the separation column,
The sample separated into each component is sprayed at approximately the speed of sound to be sent to a sonic spray ion source that is ionized, and this ion source is operated in the negative ion measurement mode, so that the neutral sugar chain in the sample and Ionize acidic sugar chains ,
A liquid chromatograph mass spectrometric method comprising mass spectrometry of an ionized sample.   2. The liquid chromatograph mass spectrometric method according to claim 1, wherein the separation column is a reverse phase column. 2. The liquid chromatograph mass spectrometry method according to claim 1, wherein the separation column is a silica column, and the eluent is a mixed solution of an aqueous ammonium acetate solution and an aqueous acetonitrile solution, A liquid chromatograph mass spectrometric method characterized by feeding the sample to a separation column while changing the mixing ratio with an aqueous acetonitrile solution. a liquid feed pump for mixing and feeding a plurality of eluents having different pHs so as to have a pH of 3 to 5;
Sample introduction means for introducing a sample containing a neutral sugar chain and an acidic sugar chain into the eluent fed by the liquid feed pump;
A separation column for separating the sample sent by the eluent into each component;
A sonic spray ion source that ionizes neutral sugar chains and acidic sugar chains in the sample by spraying the sample separated by the separation column at a substantially sound speed ;
Mass spectrometry means for mass-analyzing a sample ionized by the ion source;
A liquid chromatograph mass spectrometer comprising: 5. The liquid chromatograph mass spectrometer according to claim 4, wherein the separation column is a reverse phase column. 5. The liquid chromatograph mass spectrometer according to claim 4, wherein the separation column is a silica column, the eluent is a mixed solution of an ammonium acetate aqueous solution and an acetonitrile aqueous solution, and the liquid feed pump is the mixed solution. A liquid chromatograph mass spectrometer characterized by feeding the mixed solution while changing the mixing ratio of the aqueous ammonium acetate solution and the aqueous acetonitrile solution.

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