JPH09210983A - Chromatogram processing method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To easily make comparison of the peaks originating from the same component even in the case many peaks exist by furnishing a peak identifying means to identify the peak originating from the same component among peaks existing in the chromatogram, and providing a means to turn the difference in the area or height calculated into histogram for each peak. SOLUTION: A peak identifying means 4 has a peak sensing part 3 to sense the peak in a chromatogram prepared by a chromatogram preparing part 2 and identifies the components of the peak from the elution time of the sensed peak. An increment/decrement calculation part 5 determines the values of area and height of the peaks in different chromatograms from the identifying results and determines the difference between the peaks originating from the same component. A histogram preparing part 6 prepares histograms on the basis of given increment/decrement values.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an apparatus and method for processing a chromatogram obtained by chromatography such as high performance liquid chromatography (HPLC).

[0002]

2. Description of the Related Art In chromatography such as HPLC, the degree of the output value of a detector with respect to the elapsed time from the injection of a sample is obtained as a chromatogram. For example, when injecting a protein mixture into a column for molecular weight fractionation,
After injection, a high molecular weight protein elutes from the column at a relatively early time, and a low molecular weight protein elutes at a relatively late time. When the elution of protein from this column is detected by the change in absorbance in the ultraviolet region, the chromatogram is represented by the change in absorbance in the ultraviolet region with respect to time.

In such chromatographic analysis work, it has been practiced to see the difference between the two chromatograms. In this case, the two chromatograms are visually overlapped and the degree of change is observed. Or, generally, the areas and heights of the peaks contained in both chromatograms are digitized and compared.

In the data processing of a peptide sequencer used for determining the amino acid sequence of a protein or peptide, for example, visual analysis is generally performed as in the above. In particular, in determining the amino acid sequence, in order to clarify the degree of increase and decrease of peaks, the area and height of peaks, yield, etc. are quantified and then histogrammed, and the first chromatogram and A so-called difference chromatogram method for calculating the difference between the second chromatograms may be used.

[0005]

When looking at the difference between two chromatograms, when the number of peaks contained therein is small, it is possible to easily compare visually the magnitude of peaks derived from the same component. In addition, when observing the increase or decrease of peaks derived from the same component in two chromatograms, even when the target peak is extremely larger than the other peaks or the degree of increase or decrease is large, It is easy to judge. In such a case, it is particularly easy to calculate and compare the area value or height value of the target peak.

However, when the number of peaks is large, the target peak is extremely smaller than the other peaks, or when the degree of increase or decrease of the target peak is subtle, a histogram is visually or calculated from the calculated values. It is difficult to compare the two by creating the etc.

[0007]

The present invention is an apparatus which has been made to solve the above problems, and converts a chromatogram signal from a chromatograph into digital data by an A / D converter, and fetches the data. A first chromatogram and a first chromatogram to be compared with a first chromatogram in a data processing device for a chromatograph that executes a predetermined analysis process and stores and / or displays the chromatogram and the analysis result in a storage means. A peak identifying means for identifying a peak derived from the same component from the peaks present in the chromatogram of No. 2, a first calculating means for calculating an area value of the identified peak or a height value from the baseline; 1st and 2nd
Second calculation means for calculating, for each peak, a difference in area value or height value of peaks derived from the same component in the chromatogram, and for each peak, a difference in calculated area value or height value. A third data processing device for a chromatograph having a third calculation means for forming a histogram.

Further, according to the present invention, a chromatogram signal from a chromatograph is converted into digital data by an A / D converter and taken in, a predetermined analysis process is executed, and the chromatogram and the analysis result are stored in a storage means. And / or displaying a chromatographic data processing method, identifying a peak derived from the same component from the peaks present in the first chromatogram and the second chromatogram to be compared with the first chromatogram, Calculate the area value of the identified peak or the height value from the baseline, and calculate the difference between the peak area value or the height value derived from the same component in the first and second chromatograms for each peak. A method for processing data for a chromatograph, characterized in that the calculated difference in area value or difference in height value is histogrammed for each peak. Hereinafter, the present invention will be described in detail.

FIG. 1 is a block diagram showing an operating procedure inside the apparatus of the present invention. Reference numeral 1 is a data storage unit that records the detection signal of the detector of the chromatograph, and 2 is a chromatogram creation unit that creates a chromatogram from the data stored in the data storage unit. The peak identifying means in the present invention comprises a peak detecting section 3 for detecting the peak of the chromatogram created in 2, and a peak identifying section 4 for identifying the component of the peak from the elution time of the peak detected in 3. .

Reference numeral 5 denotes the first and second calculation means of the present invention, and the area value and height value of the peak in each chromatogram are obtained from the identification result obtained in 4, and the difference between the peaks derived from the same component is obtained. Is an increase / decrease calculation unit that calculates Reference numeral 6 is a third calculation means in the present invention, which is a histogram creation unit for creating a histogram based on the increase / decrease value obtained in 5.

Numerals 7 and 8 are portions responsible for functions preferably added to the device of the present invention. Reference numeral 7 is an increase / decrease level dividing unit that divides the histogram created in 6 into levels according to the degree thereof, and 8 is the histogram obtained in 6 and 7
This is an output unit that outputs the level-divided values and other calculation results obtained in.

In liquid chromatography and the like, a chromatogram is created from the detection signal of the detector obtained at each time. First, a sample with known components is analyzed, and the elution time of each component is measured to identify it. Obtain the data (chromatogram) for. Next, an unknown sample used under the same conditions is analyzed to obtain a chromatogram. Peaks are detected by using the obtained chromatograms as the first and second chromatograms, for example, the peaks having the same elution time are identified as the peaks derived from the same component.

For example, when the following results are obtained for the first and second chromatograms, the increase / decrease in the peak of chromatogram 2 with respect to chromatogram 1 is calculated from the calculation result of chromatogram 2 to chromatogram 1 It can be calculated by subtracting the calculation result of.

* Peak detection result of chromatogram 1 Number Name Time Height Area 1 N1 T11 H11 A11 2 N2 T12 H12 A12 3 N3 T13 H13 A13 :: :: :: :: :: :: :: (m-1) N (m -1) T1 (m-1) H1 (m-1) A1 (m-1) m Nm T1m H1m A1m * Peak detection result of chromatogram 2 No. Name Time Height Area 1 N1 T21 H21 A21 2 N2 T22 H22 A22 3 N3 T23 H23 A23 :::::::::::: (m-1) N (m-1) T2 (m-1) H2 (m-1) A2 (m-1) m Nm T2m H2m A2m * Difference in chromatogram 2-1 No. Name Height Area 1 N1 H21-H11 A21-A11 2 N2 H22-H12 A22-A12 3 N3 H23-H13 A23-A13 :::::::: (m-1) N (m-1) H2 (m-1) -H1 (m-1) A2 (m-1) -A1 (m-1) m Nm H2m-H1m A2m-A1m Next, the result obtained as above Is made into a histogram, and the display items in the histogram are the first and second
Is the peak difference area value and / or the peak difference height value of the two chromatograms. In the present invention, in addition to these, the peak area value and / or height value of both and / or one of the two chromatograms may be displayed, if necessary.
It is also possible to display, for each peak, the name of the substance that generated the peak and the like.

For example, by taking a peak name on one of the X-axis and Y-axis of the histogram and a value on the other axis, the area value, the difference in area value, the peak height value, the difference in peak height value, etc. are made into a histogram. indicate. An example of the display result in the present invention is shown in FIGS. FIG. 2 shows the peak name on the X-axis and the peak height difference on the Y-axis. FIG. 3 shows the peak name on the Y-axis and the peak height difference on the X-axis. Of course, in the present invention, the difference in peak area may be displayed instead of the difference in peak height.

In a peptide sequencer for identifying an amino acid sequence, an amino acid derivative is first produced and subjected to high performance liquid chromatography (HPLC) to obtain a chromatogram of each residue as an analysis result. Next, when identifying the amino acid of each residue corresponding to the chromatogram peak, the increased peak and the decreased peak in the obtained chromatogram are read to determine the amino acid. That is, the amino acid of a certain residue (n residue) is compared with the chromatogram of the immediately preceding residue (n-1 residue), and the greatly increased peak is judged to be the amino acid of the n residue. In addition, in comparison with the chromatogram of the n-1 residue, the peak greatly reduced is determined to be the amino acid of the (n-1) residue. In other words, in a sequencer having an amino acid sequence, an operation of comparing two chromatograms is essential, so that the present invention can be preferably applied.

In the present invention, preferably, the degree of increase or decrease of the target peak can be classified into levels based on the difference in peak height or the difference in peak area, and can be used as an index for chromatogram judgment.

For example, a mark indicating an increase can be added to the increased peak according to the ratio. That is, the increased peaks are extracted from the identified peaks in the chromatogram, and the sum of them is calculated. Then, for each increased peak, the ratio (%) to the total sum is calculated, and the marks are distributed according to the ratio. As an example, when the rate of increase is 100% to 80%, "++++", and when it is 80% to 50%, "++", 50
For example, a mark "+" may be added in the case of 30% to 30%, and a "blank" mark may be added in the case of 30% to 0%.

On the other hand, it can be illustrated that a mark is similarly added to the reduced peak. That is, the reduced peaks are extracted from the identified peaks in the chromatogram, and the total sum is calculated. Then, for each reduced peak, the ratio (%) to the total sum is calculated, and the marks are distributed according to the ratio. As an example, when the reduction rate is 100% to 80%, "---", 80% to 5
For example, a mark of "-" may be added when 0%, a mark of "-" when 50% to 30%, and a mark of "blank" when 30% to 0%.

An example of a calculation formula for assigning a mark to an increased or decreased peak in the above-mentioned amino acid sequencer is shown below.

* Marking of increased peaks Ii: increase rate of peak height of amino acid Hi: peak height of increased amino acid n: number of increased amino acid 80 ≦ Ii ≦ 100 then Mark = ++++ 50 ≦ Ii <80 then Mark = +++ 30 ≦ Ii <50 then Mark = + 0 ≦ Ii <30 then Mark = Blank * Marking to reduced peak Di: Peak height reduction rate of amino acids HD: Peak height of reduced amino acids n: Number of reduced amino acids 80 ≦ Ii ≦ 100 then Mark = −−− 50 ≦ Ii <80 then Mark = −− 30 ≦ Ii < 50 then Mark = −0 ≦ Ii <30 then Mark = blank Although an example of the calculation method regarding the increase or decrease of the peak height is shown here, the peak area value can be treated in the same manner.

The present invention can be preferably applied to a peptide sequencer for identifying an amino acid sequence. The flow of the sequencer processing based on the present invention is shown in FIG. First, Edman degradation is performed with a peptide sequencer to generate an amino acid derivative. The produced amino acid derivative is separated by HPLC, and its detector signal is stored in a data storage medium.

Next, from the chromatogram of the nth residue, (n
-1) Subtract the residue chromatogram to calculate the difference in peak area or the difference in peak height. The level is divided from the results obtained as necessary, and then the peak area value and / or peak height value of the chromatogram of the n-th residue for each amino acid, the difference in peak area value, and / or the peak height The difference between the height values is converted into a histogram and output to a CRT, a printer or the like.

As the peptide sequencer based on the present invention, for example, one having a structure as shown in FIG. 5 can be exemplified. This device is composed of a sequencer section (18), an HPLC section (11 to 15), and a data processing section (16, 17).

The HPLC section comprises a pump (12), a sample injector (13), an analytical column (14) and a detector (15). The eluent is sent to the analytical column by the pump (12) at 200 μl / min. As the analytical column, a commercially available reverse-phase packing material (TSKgel PTHpak, manufactured by Tosoh Corporation) was used, and the detector was set to an ultraviolet-visible detector at 269 nm.

The PTH-amino acid produced by the peptide sequencer is pressure-fed to the injector (13) under nitrogen gas pressure, and a predetermined amount is injected into the analytical column for separation and detection. The data processing section performs processing such as peak detection on the signal from the detector (15), and the result is the data storage medium (17).
Is stored in

[0027]

EXAMPLES Examples will be described below in order to explain the present invention in more detail, but the present invention is not limited to these examples.

Example 1 Using the peptide sequencer having the configuration shown in FIG. 5, β-lactoglobulin sequence measurement was carried out under the above-mentioned conditions. The results are shown in FIGS. 6 to 8.

FIG. 6 is a chromatogram of the second residue,
FIG. 7 is a chromatogram of the third residue. FIG. 8 shows the chromatogram of the third residue with respect to the chromatogram of the second residue.
The result (histogram) obtained by applying the present invention is shown.

In FIG. 8, the peak height is used as a criterion to make an increase or decrease determination. In the figure, the X axis is the peak name (the name of the amino acid residue that generated each peak), Y
The axis is the peak height value in the histogram of the third residue and the difference between the peak height values of the chromatogram of the third residue and the chromatogram of the second residue (white histogram was detected in the three residues. (A peak height value of amino acid, a value obtained by subtracting the peak height value of the amino acid detected at 2 residues from the peak height value of the amino acid detected at 3 residues in the black histogram). In the figure, it is clearly seen that the increase of amino acids marked Val and the decrease of amino acids marked Ile are remarkable.

The judgment shown in FIG. 8 is added as described above. In the histogram of FIG. 8, the increased peaks are Asn, Gln, Ser, As.
p, Glu, Gly, Ala, Tyr, His, Me
For t, Val, and Phe, the total of the detector output signals (total height difference of chromatogram) was 26.80 mV. The difference in peak height derived from Val is 24.44.
Since it is mV, the increase rate is 24.44 × 100/2
6.80 = 90.37%, and the mark of “++++” was added according to the criteria as described above.

The reduced peaks are Thr, Arg and Pr.
o, DPTU, Ile, and Leu, the total detector output signal (total chromatogram height difference) is 18.6.
It was 1 mV. The peak height difference due to Ile is 1
Since it is 4.16, the reduction rate is 14.16 × 100.
/18.61=76.09%, and the mark "---" was added according to the criteria as described above.

From the marks showing the increase and decrease obtained in this way, the increase in the amino acid marked Val is remarkable, and Ile
It can be intuitively understood that the decrease in the amino acids marked with is marked, and from the histogram in FIG. 8 and the added marks, 3
The residue amino acid is Val, the second residue amino acid is Il
was estimated to be e.

[0034]

As is apparent from the above description, according to the present invention, when the number of peaks is large, the target peak is extremely smaller than the other peaks, and the degree of increase or decrease of the target peak is further increased. Even if is subtle, it is possible to easily compare peaks derived from the same component.

The present invention is particularly effective for a peptide sequencer whose main purpose is to estimate an amino acid sequence based on the increase / decrease of peaks. That is, according to the present invention, it is possible to intuitively identify an amino acid, and it is possible to obtain a highly sensitive and stable result even in amino acid sequence analysis in a dilute sample.

[Brief description of drawings]

FIG. 1 is a block diagram showing an operating procedure inside an apparatus of the present invention.

FIG. 2 is a diagram showing an example of a histogram according to the present invention.

FIG. 3 is a diagram showing an example of a histogram according to the present invention.

FIG. 4 is a block diagram for explaining the peptide sequencer of the present invention.

FIG. 5 is a diagram showing an example of the configuration of the peptide sequencer of the present invention.

FIG. 6 shows the results of the examples of the present invention, and is a chromatogram for the second residue of the Edman degradation product of β-lactoglobulin. In the figure, the horizontal axis represents the elution time (minutes), and the vertical axis represents the output signal intensity of the UV-visible detector, that is, the peak height value (mV). The numbers attached to the peaks in the figure indicate the peak names, and are 1; As, respectively.
n, 2; Gln, 3; Asp, 4; Thr, 5; Gl
u, 6; Ala, 7; Tyr, 8; His, 9; Ar
g, 10; Met, 11; Val, 12; Pro, 1
3; DPTU, 14; Phe, 15; Ile, 16; L
eu (amino acid residues are represented by the usual three letters of the alphabet). The lower part of the figure shows the retention time (min) of each peak name, the output signal strength of the detector, that is, the peak height value (mV).

FIG. 7 shows the results of the examples of the present invention, and is a chromatogram for the third residue of the Edman degradation product of β-lactoglobulin. In the figure, the horizontal axis represents the elution time (minutes), and the vertical axis represents the output signal intensity of the UV-visible detector, that is, the peak height value (mV). The numbers attached to the peaks in the figure indicate the peak names, and are 1; As, respectively.
n, 2; Gln, 3; Ser, 4; Asp, 5; Th
r, 6; Glu, 7; Gln, 8; Ala, 9; Ty
r, 10; His, 11; Arg, 12; Met, 1
3; Val, 14; Pro, 15; DPTU, 16; P
he, 17; Ile, 18; Leu (the amino acid residue is
Indicated by the usual three letters). The lower part of the figure shows the retention time (min) of each peak name, the output signal strength of the detector, that is, the peak height value (mV).

[Fig. 8] Fig. 8 shows the results of the examples of the present invention. The chromatogram for the third residue of the Edman degradation product of β-lactoglobulin obtained by the method of the present invention shows 2
It is a histogram obtained by subtracting the chromatograms related to the residues. In the figure, the horizontal axis represents the name of the amino acid residue causing each peak, and the vertical axis represents the output signal intensity of the UV-visible detector, that is, the peak height value (mV). In the figure,
Amino acid residues are represented by the usual three letters of the alphabet. The lower part of the figure shows the peak number, the name of the amino acid residue that caused the peak, the elution time (minutes), the output signal intensity of the detector in FIG. 7 (peak height value (mV)), and FIG. 6 shows the difference (mV) between the detector output signal intensity (peak height value (mV)) and the detector output signal intensity (peak height value (mV)) in FIG. 6 and the determination described above. It is a thing.

[Explanation of symbols]

 1 Data Storage Section 2 Chromatogram Creation Section 3 Peak Detection Section 4 Peak Identification Section 5 Increase / Decrease Calculation Section 6 Histogram Creation Section 7 Increase / Decrease Level Division Section 8 Output Section 11 Eluent 12 Pump 13 Sample Injector 14 Separation Column 15 Detector 16 Data processor 17 Data storage medium 18 Peptide sequencer

Claims (6)

[Claims] 1. A chromatogram signal from a chromatograph is converted into digital data by an A / D converter and fetched, a predetermined analysis process is executed, and the chromatogram and the analysis result are stored in a storage means and / or. In a chromatograph data processing device for displaying, a peak identifying means for identifying a peak derived from the same component from peaks present in a first chromatogram and a second chromatogram to be compared with the first chromatogram. A first calculation means for calculating the area value or the height value from the baseline of the identified peak, and the area value or the height value of the peak derived from the same component in the first and second chromatograms. There is provided a second calculation means for calculating the difference for each peak and a third calculation means for making a histogram of the calculated difference in area value or difference in height value for each peak. Chromatograph data processing device. 2. The chromatograph data processing apparatus according to claim 1, further comprising fourth calculating means for classifying the difference between the area value or the height value calculated by the second calculating means. 3. The chromatograph data processing according to claim 1, further comprising a storage / display device for storing and / or displaying a result calculated by the third calculating means and / or the fourth calculating means. apparatus. 4. A liquid chromatography apparatus comprising the chromatographic data processing apparatus according to claim 1. 5. A peptide sequencer device comprising the chromatographic data processing device according to claim 1 and utilizing sequential terminal amino acid excision of a protein or peptide by Edman degradation reaction. 6. A chromatogram signal from a chromatograph is converted into digital data by an A / D converter and fetched, a predetermined analysis process is executed, and the chromatogram and the analysis result are stored in a storage means and / or. In the displayed data processing method for a chromatograph, a peak derived from the same component was identified and identified from the peaks present in the first chromatogram and the second chromatogram to be compared with this first chromatogram. The peak area value or the height value from the baseline is calculated, and the difference between the peak area value or the height value derived from the same component in the first and second chromatograms is calculated for each peak. A method for processing data for a chromatograph, characterized in that a difference in area value or a difference in height value is converted into a histogram for each peak.