METHOD FOR ANALYZING METALLOPROTEIN IN BIOLOGICAL SAMPLE

Abstract

The present invention relates to a method for analyzing a metalloprotein in a biological sample capable of continuously maintaining conditions of LC-ICPMS constant to measure a metalloprotein with high data reliability. The method for analyzing a metalloprotein which is a complex in a biological sample, the metalloprotein being a complex in which a biomolecule and a metal element bind to each other, includes: treating a biological sample which has been subjected to a pretreatment by liquid chromatography to separate the metalloprotein, and analyzing the separated metalloprotein by inductively coupled plasma mass spectrometry, wherein an ammonium acetate solution is used as a mobile phase.

Claims

1. A method for analyzing a metalloprotein in a biological sample, the metalloprotein being a complex in which a biomolecule and a metal element bind to each other, the method comprising: treating the biological sample that has been subjected to a pretreatment by liquid chromatography to separate the metalloprotein; detecting the separated metalloprotein by a UV detector; and analyzing the separated metalloprotein by inductively coupled plasma mass spectrometry after detecting the separated metalloprotein by the UV detector, wherein an ammonium acetate solution is used as a mobile phase.

2. The method for analyzing a metalloprotein in a biological sample as recited in claim 1, wherein the liquid chromatography is size exclusion chromatography.

3. (canceled)

4. The method for analyzing a metalloprotein in a biological sample as recited in claim 1, wherein a concentration of the ammonium acetate solution is 25 mM to 100 mM.

5. The method for analyzing a metalloprotein in a biological sample as recited in claim 2, wherein a pH value of the ammonium acetate solution is between 6 and 7.

6. The method for analyzing a metalloprotein in a biological sample as recited in claim 1, wherein immunoaffinity chromatography is used in the pretreatment.

7. The method for analyzing a metalloprotein in a biological sample as recited in claim 1, wherein the metal element is K, P, Na, Ca, Mg, Al, As, Hg, Pb, Cd, Ti, Ag, Ba, Zn, Cr, Mn, Cu, Rb, Fe, Ge, Se, Sr, Co, Ni, Mo, Sn, Sb, Pt, Cs, U, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Y, Li, or B.

8. The method for analyzing a metalloprotein in a biological sample as recited in claim 1, wherein the separated metalloprotein is further analyzed by electrospray ionization mass spectrometry when analyzing by the inductively coupled plasma mass spectrometry.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] (a) to (d) of FIG. 1 are chromatograms of two elements, Fe and Co, measured in Example 1.

[0021] FIG. 2 is a chromatogram of specified 47 types of elements measured in Example 2.

[0022] FIG. 3 is a chromatogram of specified 47 types of elements measured in Example 2.

[0023] FIG. 4 is a chromatogram of specified 47 types of elements measured in Example 2.

[0024] FIG. 5 is a chromatogram of specified 47 types of elements measured in Example 2.

[0025] FIG. 6 is a chromatogram of specified 47 types of elements measured in Example 2.

[0026] FIG. 7 is a chromatogram of specified 47 types of elements measured in Example 2.

[0027] FIG. 8 is a chromatogram of specified 47 types of elements measured in Example 2.

[0028] FIG. 9 is a chromatogram of specified 47 types of elements measured in Example 2.

[0029] FIG. 10 is a chromatogram of specified 47 types of elements measured in Example 2.

[0030] FIG. 11 is a chromatogram of specified 47 types of elements measured in Example 2.

[0031] FIG. 12 is a chromatogram of specified 47 types of elements measured in Example 2.

[0032] FIG. 13 is a chromatogram of specified 47 types of elements measured in Example 2.

[0033] FIG. 14 is a chromatogram of specified 47 types of elements measured in Example 2.

[0034] FIG. 15 is a chromatogram of specified 47 types of elements measured in Example 2.

[0035] FIG. 16 is a chromatogram of specified 47 types of elements measured in Example 2.

[0036] FIG. 17 is a chromatogram of specified 47 types of elements measured in Example 2.

[0037] FIG. 18 is a chromatogram of specified 47 types of elements measured in Example 2.

[0038] FIG. 19 is a chromatogram of specified 47 types of elements measured in Example 2.

[0039] FIG. 20 is a chromatogram of specified 47 types of elements measured in Example 2.

[0040] FIG. 21 is a chromatogram of specified 47 types of elements measured in Example 2.

[0041] FIG. 22 is a chromatogram of specified 47 types of elements measured in Example 2.

[0042] FIG. 23 is a chromatogram of specified 47 types of elements measured in Example 2.

[0043] FIG. 24 is a chromatogram of specified 47 types of elements measured in Example

[0044] FIG. 25 is a chromatogram of specified 47 types of elements measured in Example

[0045] FIG. 26 is a chromatogram of specified 47 types of elements measured in Example

[0046] FIG. 27 is a chromatogram of specified 47 types of elements measured in Example

[0047] FIG. 28 is a chromatogram of specified 47 types of elements measured in Example

[0048] FIG. 29 is a chromatogram of specified 47 types of elements measured in Example

[0049] FIG. 30 is a chromatogram of specified 47 types of elements measured in Example

[0050] FIG. 31 is a chromatogram of specified 47 types of elements measured in Example

[0051] FIG. 32 is a chromatogram of specified 47 types of elements measured in Example

[0052] FIG. 33 is a chromatogram of specified 47 types of elements measured in Example

[0053] FIG. 34 is a chromatogram of specified 47 types of elements measured in Example

[0054] FIG. 35 is a chromatogram of specified 47 types of elements measured in Example

[0055] FIG. 36 is a chromatogram of specified 47 types of elements measured in Example 2.

[0056] FIG. 37 is a chromatogram of specified 47 types of elements measured in Example 2.

[0057] FIG. 38 is a chromatogram of specified 47 types of elements measured in Example 2.

[0058] FIG. 39 is a chromatogram of specified 47 types of elements measured in Example 2.

[0059] FIG. 40 is a chromatogram of specified 47 types of elements measured in Example 2.

[0060] FIG. 41 is a chromatogram of specified 47 types of elements measured in Example 2.

[0061] FIG. 42 is a chromatogram of specified 47 types of elements measured in Example 2.

[0062] FIG. 43 is a chromatogram of specified 47 types of elements measured in Example 2.

[0063] FIG. 44 is a chromatogram of specified 47 types of elements measured in Example 2.

[0064] FIG. 45 is a chromatogram of specified 47 types of elements measured in Example 2.

[0065] FIG. 46 is a chromatogram of specified 47 types of elements measured in Example 2.

[0066] FIG. 47 is a chromatogram of specified 47 types of elements measured in Example 2.

[0067] FIG. 48 is a chromatogram of specified 47 types of elements measured in Example 2.

[0068] (a) to (d) of FIG. 49A are chromatograms of two elements, Fe and Co, measured at varying concentrations of the mobile phase in Example 3.

[0069] (e) to (h) of FIG. 49B are chromatograms of two elements, Fe and Co, measured at varying concentrations of the mobile phase in Example 3.

[0070] (i) to (l) of FIG. 49C are chromatograms of two elements, Fe and Co, measured at varying concentrations of the mobile phase in Example 3.

[0071] (a) and (b) of FIG. 50 are analytical diagrams showing the consecutive operation results of LC-ICPMS in Example 4.

[0072] (a) and (b) FIG. 51A show LC-UV-ICPMS chromatograms of a standard sample of Example 4 and the calibration curve.

[0073] (c) to (h) of FIG. 51B show LC-UV-ICPMS chromatograms of a standard sample of Example 4 and the calibration curve.

[0074] (i) to (n) of FIG. 51C show LC-UV-ICPMS chromatograms of a standard sample of Example 4 and the calibration curve.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

[0075] The method for analyzing a metalloprotein in a biological sample according to the present invention is a method that performs an analysis using LC-ICPMS in the same manner as in prior art. In order to avoid blockage of a skimmer by deposited solids, a volatile ammonium acetate solution is used as the mobile phase. With this, in the present invention, it is possible to continuously analyze a biological sample without requiring frequent apparatus maintenance and maintain the condition of the analysis constant to ensure data reliability.

[0076] Specifically, according to the analysis method of this embodiment, initially, a pretreatment is performed on a biological sample. In the pretreatment, it is preferable to remove several types of proteins high in content, such as, e.g., albumin, IgG, transferrin, IgA, haptoglobin, and antitrypsin, by immunoaffinity chromatography. These macromolecule proteins are often indistinguishable from metalloproteins by SEC (size exclusion chromatography), which will be described later. The use of immunoaffinity chromatography enables the avoidance of interference by these proteins.

[0077] Thereafter, the biological sample that has been subjected to the pretreatment is processed to separate the metalloprotein by liquid chromatography. The use of the size exclusion chromatography (SEC) enables the separation of metalloproteins and free metal elements.

[0078] Next, the separated metalloproteins are analyzed by inductively coupled plasma mass spectrometry (ICPMS) to determine the type and content. In this embodiment, it is preferred to further detect metalloproteins after LC by a UV detector before performing the analysis by ICPMS. As an advantage to do this, in cases where the detection signal of the ICPMS is abnormally attenuated, by comparing the detection signal of the UV detector with the detection signal of the inductively coupled plasma mass spectrometry (ICPMS), it is possible to determine whether the liquid chromatography (LC) is failed or the inductively coupled plasma mass spectrometry (ICPMS) is failed, e.g., whether the skimmer is blocked. For example, in cases where the detection result of the UV detector is normal and the detection result of the inductively coupled plasma mass spectrometry (ICPMS) is obviously attenuated, it can be determined that it is a failure of the inductively coupled plasma mass spectrometry (ICPMS). On the other hand, in cases where both the detection result of the UV detector and that of the inductively coupled plasma mass spectrometry (ICPMS) are abnormally attenuated or not detected, it can be determined that the failure of the liquid chromatography (LC).

[0079] Note that it is also possible to analyze the separated metalloprotein using electrospray ionization mass spectrometry (ESIMS) when analyzing the metalloprotein separated by inductively coupled plasma mass spectrometry (ICPMS) at the same time. The analysis by the inductively coupled plasma mass spectrometry (ICPMS) reveals which types of metal elements are contained in this metalloprotein. The electrospray ionization mass spectrometry (ESIMS) can measure the molecular weight of this metalloprotein. The combination thereof enables to specify this metalloprotein.

[0080] Note that performing the size exclusion chromatography (SEC) under mild conditions (close to the biological environment) not only maintains the protein in its native state but also provides the approximate molecular size information. Therefore, in the liquid chromatography (LC), the pH value of the ammonium acetate solution is preferably 6 to 7.

[0081] The feasibility and the data reliability when using an ammonium acetate solution as a mobile phase in a method for analyzing a metalloprotein LC-ICPMS will be described by exemplifying four examples.

Example 1

[0082] In this Example, two elements of Fe and Co were measured, and the concentration of the mobile phase was set to 100 mM.

[0083] As the biological sample, a mixed solution of a commercially available sample A: SIGMA Human Serum H4522 (containing Fe elements) and a commercially available sample B: Wako Cyanocobalamin, C63H88CoN14O14P, MW 1355. 38 (vitamin B12, VB12) (containing Co elements), SIGMA Myoglobin (obtained from horse hearts), and MW 17 kDa (containing Fe elements), was adopted. All parts which come into contact with the solution were made of metal-free materials.

[0084] The LC-ICPMS analysis conditions of this Example are shown in Tables 1-1 and Table 1-2 shown below.

Table 1

[0085]

TABLE-US-00001 TABLE 1-1 Apparatus used Apparatus Shimadzu High-Speed liquid chromatograph LC-20 Inert used: High-Pressure GE System System Controller CBM-20Alite Liquid feed unit LC-20Ai On-line deaeration unit DGU-20A.sub.5R Auto-sampler SIL-20AC + SIL inert kit Column-oven CTO-20AC Mixer Mixer PEEK 1.6 mL UV detector SPD-20AV + semi micro cell ICPMS ICPMS-2030 LC-workstation Lab Solutions Ver. 5.82 sp1 ICPMS Workstation Lab Solutions ICPMS TRM Ver. 1.02

TABLE-US-00002 TABLE 1-2 LC-ICPMS Analysis Conditions Column Phenomenex Yarra SEC-X300 (300 mmL. 4.6 mm I.D., 1.8 m) Mobile phase 100 mmol/L aqueous ammonium acetate solution, near pH 6 Mobile phase flow 0.3 mL/min (pressure 18.2 MPa) rate Column temperature 35 C. Injection volume 10 L (SIL wash solution: water) Sample SIGMA Human Serum H4522-100ML All samples were filtered and diluted 2 times with 10 mM ammonium acetate-water Wako Cyanocobalamin, C63H88CoN14O14P, MW 1355.38 (Vitamin B12, VB12) SIGMA Myoglobin from equine heart, MW 17 kDa Dissolve in the mobile phase Detector Shimadzu UV detector SPD-20AV Semi-micro temperature control cell Lamp: D.sub.2 Wavelength: 210, 280 nm Sampling: 2 Hz Cell temperature: 40 C. Capture time: 21 min ICPMS See FIG. 1-1 Capture time: 20 min Detected ion: Dwell time element m/z (sec) Fe 56 0.1 Co 59 0.1 Measurement time 21 min

[0086] The results of the analyses are shown in FIG. 1. (a) of FIG. 1 shows the signal of the metalloprotein when the sample A was subjected to liquid chromatography (LC). The signal was detected by a UV detector with the sample irradiated with 280 nm light. (b) of FIG. 1 shows the signal of the metalloprotein when the sample A was subjected to liquid chromatography (LC). The signal was detected by inductively coupled plasma mass spectrometry (ICPMS). (c) of FIG. 1 shows the signal of the metalloprotein when the sample B was subjected to liquid chromatography (LC). The signal was detected by a UV detector with the sample irradiated with 280 nm light. (d) of FIG. 1 shows the signal of the metalloprotein when the sample B was subjected to liquid chromatography (LC). The signal was detected by inductively coupled plasma mass spectrometry (ICPMS).

[0087] The two peaks in (b) of FIG. 1 show proteins containing two types of irons, and no protein containing cobalt was detected. The two peaks in (c) of FIG. 1 indicate two components in the sample B. The two peaks in (d) of FIG. 1 indicate the metal elements Fe and Co.

Example 2

[0088] LC-ICPMS (Liquid chromatographyInductively coupled plasma mass spectrometry) using an ammonium acetate solution as a mobile phase can analyze all known types of metalloproteins. In this Example, 47 types of metal elements among them were analyzed and measured, but the measurable ranges are not limited thereto. The sample A is adopted as a biological sample, and the concentration of the mobile phase was set to 100 mM.

[0089] The apparatus used in this Example is the same as that used in Example 1. The analysis conditions of LC-ICPMS are shown in Table 2 shown below.

TABLE-US-00003 TABLE 2 Column Phenomenex Yarra SEC-X300 (300 mmL. 4.6 mm I.D., 1.8 m) Mobile phase 100 mmol/L Aqueous ammonium acetate solution, near pH 6 Mobile phase flow 0.3 mL/min (pressure 18.2 MPa) rate Column temperature 35 C. Injection volume 10 L (SIL wash solution: water) Sample SIGMA Human Serum H4522-100ML All samples were filtered and diluted 2 times with 10 mM ammonium acetate-water Detector Shimadzu UV detector SPD-20AV Semi-micro temperature control cell Lamp: D.sub.2 Wavelength: 210, 280 nm Sampling: 2 Hz Cell temperature: 40 C. Capture time: 21 min ICPMS Capture time: 20 min Detected ion: element K, P, Na, Ca, Mg, Al, As, Hg, Pb, Cd, Ti, Ag, Ba, Zn, Cr, Mn, Cu, Rb, Fe, Ge, Se, Sr, Co, Ni, Mo, Sn, Sb, Pt, Cs, U, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Y, Li, B. Dwell time (sec) each 0.05 Measurement time 21 min

[0090] ICPMS analysis results of these 47 elements are shown in FIG. 2 to FIG. 48.

Example 3

[0091] In this Example, two elements of Fe and Co were measured. As biological samples, the sample A and the sample B were adopted, respectively, and the concentrations of the mobile phases were set to 100 mM, 50 mM, and 25 mM, respectively.

[0092] The apparatus used in this Example was the same as that used in Example 1. LC-ICPMS analytical conditions are shown in Table 3 shown below.

TABLE-US-00004 TABLE 3 Column Phenomenex Yarra SEC-X300 (300 mmL. 4.6 mm I.D., 1.8 m, MW range 10k-700 kDa) Mobile phase 100 mmol/L aqueous ammonium acetate solution, near pH 6 50 mmol/L aqueous ammonium acetate solution, near pH 6 25 mmol/L aqueous ammonium acetate solution, near pH 6 Mobile phase flow 0.3 mL/min (pressure 18.2 MPa) rate Column temperature 35 C. Injection volume 10 L (SIL wash solution: water) Sample SIGMA Human Serum H4522-100ML All samples were filtered and diluted 2 times with 10 mM ammonium acetate-water and Wako Cyanocobalamin, C63H88CoN14O14P, MW 1355.38 (Vitamin B12, VB12) SIGMA Myoglobin from equine heart, MW 17 kDa Dissolve in the mobile phase Detector Shimadzu UV detector SPD-20AV Semi-micro temperature control cell Lamp: D.sub.2 Wavelength: 210, 280 nm Sampling: 2 Hz Cell temperature: 40 C. Capture time: 21 min ICPMS See FIG. 2-2 Capture time: 20 min Detected ion: Dwell time element m/z (sec) Fe 56 0.100 Co 59 0.100 Measurement time 21 min

[0093] The analysis results are shown in FIG. 49. (a) to (c) of FIG. 49 show the signal of the metalloprotein when the sample A was subjected to liquid chromatography (LC). The signal was detected by the UV detector with the sample irradiated with 280 nm light. The concentrations of the mobile phases in (a) to (c) of FIG. 49 were 100 mM, 50 mM, and 25 mM, respectively. (d) to (f) of FIG. 49 show the signal of the metalloprotein when the sample A was subjected to liquid chromatography (LC). The signal was detected by ICPMS. The concentrations of the mobile phases in (d) to (f) of FIG. 49 were 100 mM, 50 mM, and 25 mM, respectively. (g) to (i) of FIG. 49 show the signal of a metalloprotein when the sample B was subjected to liquid chromatography (LC). The signal was detected by the UV detector with the sample irradiated with 280 nm light. The concentrations of the mobile phases in (g) to (i) of FIG. 49 were 100 mM, 50 mM, and 25 mM, respectively. (j) to (l) of FIG. 49 show the signal of the metalloprotein when the sample B was subjected to liquid chromatography (LC). The signal was detected by ICPMS. The concentrations of the mobile phases in (j) to (l) of FIG. 49 were 100 mM, 50 mM, and 25 mM, respectively.

Example 4

[0094] The apparatus used in this Example is the same as that used in Example 1. As described above, the use of ammonium acetate solution as the mobile phase enables continuous analyses of many samples without interruption for maintenance. In this Example, ICPMS standard samples were continuously measured over 300 times by flow injection in order to confirm the accuracy of ICPMS data when an ammonium acetate solution was used as the mobile phase. The measurement results are shown in FIG. 50. The LC-ICPMS analysis conditions of the measurements in FIG. 50 are shown in Table 4 shown below.

TABLE-US-00005 TABLE 4 Mobile phase 100 mmol/L aqueous ammonium acetate solution, near pH 6 Mobile phase flow 0.50 mL/min rate Column temperature Room temperature Injection volume 10 L (SIL wash solution: water) Sample ICPMS standard samples; Be 1 ppb, Co and Mn 0. 5 ppb, In and Bi and Ce 0.2 ppb in 1% (0.14 mol/L) HNO.sub.3 Detector Shimadzu UV detector SPD-20AV Semi-micro temperature control cell Lamp: D.sub.2 Wavelength: 210, 280 nm Sampling: 2 Hz Cell temperature: 40 C. Capture time: 15 min ICPMS See FIG. 2-2 Capture time: 14 min Detected ion: Dwell time element m/z (sec) Ce 140 0.100 Co 59 0.100 In 115 0.100 Mn 55 0.100 Measurement time 15 min

[0095] (a) of FIG. 50 shows the peak area of the ICPMS chromatogram obtained for each sample injection analysis in 300 or more analyses. The horizontal axis shows the number of injections, and the vertical axis shows the peak area. (b) of FIG. 50 shows the detection results (peak height and peak area) of the connected UV detector in these 300 or more analyses. As can be seen from FIG. 50, the data reproducibility is good by performing uninterrupted continuous measurements with an ammonium acetate solution as a mobile phase, and thus the reliability is high.

[0096] On the other hand, the sample B was measured at the analysis conditions shown in Table 5, and the peak area and the peak height of the chromatogram at different injection amounts were acquired and shown in FIG. 51. (a) and (b) of FIG. 51 show the detection results of the UV detector and the ICPMS, respectively, and (c) to (n) of FIG. 51 show the calibration curves for this standard sample, respectively.

[0097] As can be seen from FIG. 51, the peak heights and peak areas of the chromatograms detected by the ICPMS with the ultraviolet 210 nm and 280 nm, respectively, exhibit good linearity (correlation coefficient R2>0.996). Therefore, the elemental quantities can be evaluated with the peak area and peak height of LC-ICPMS chromatogram.

TABLE-US-00006 TABLE 5 Column Phenomenex Yarra SEC-X300 (300 mmL. 4.6 mm I.D., 1.8 m, MW range 10k-700 kDa) Mobile phase 100 mmol/L aqueous ammonium acetate solution, near pH 6 Mobile phase flow 0.3 mL/min (pressure 18.2 MPa) rate Column temperature 35 C. Injection volume 10 L (SIL wash solution: water) Sample Wako Cyanocobalamin, C63H88CoN14O14P, MW 1355.38 (Vitamin B12, VB12) SIGMA Myoglobin from equine heart, MW 17 kDa Dissolve in the mobile phase Detector Shimadzu UV detector SPD-20AV Semi-micro temperature control cell Lamp: D.sub.2 Wavelength: 210, 280 nm Sampling: 2 Hz Cell temperature: 40 C. Capture time: 21 min ICPMS See FIG. 2-2 Capture time: 20 min Detected ion: Dwell time element m/z (sec) Fe 56 0.100 Co 59 0.100 Measurement time 21 min