KR101748588B1 - Ovarian cancer detection method using N-glycan biomarkers - Google Patents
Ovarian cancer detection method using N-glycan biomarkers Download PDFInfo
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Abstract
The present invention relates to an oligosaccharide marker for ovarian cancer diagnosis, and more particularly, to an oligosaccharide marker for diagnosing ovarian cancer by extracting oligosaccharide at a higher rate than the conventional method while ensuring high reproducibility when a sugar protein sample derived from body fluids and tissues including serum, A method for detecting N-glycan marker for ovarian cancer diagnosis from a large amount of samples using an automated system and a high sensitivity high resolution mass spectrometer and a method for detecting N-glycosylation frequency marker and abundance marker for detecting ovarian cancer .
Description
The present invention relates to an oligosaccharide marker for ovarian cancer diagnosis, and more particularly, to an oligosaccharide marker for the diagnosis of ovarian cancer by automating the extraction of oligosaccharide at a higher rate than the conventional method, while ensuring high reproducibility when a large amount of glycoprotein- The present invention relates to a method for detecting ovarian cancer using an N-glycosylation frequency marker and an abundance marker for ovarian cancer diagnosis, which are detected from a large amount of blood samples using a high sensitivity and high resolution mass spectrometer .
Glycosylation is the most widely known process of post-translational modification (PTM), which is largely involved in the function of proteins. More than 50% of the body's proteins are glycosylated proteins. Glycoproteins are present in most body tissues as well as body fluids such as blood, saliva, and tears. The resulting oligosaccharide, which is the result of glycosylation, is very sensitive to changes in the environment outside the body, and is being used as a biomarker for various diseases including cancer. Also, in biopharmaceuticals, oligosaccharides are known to play an important role in the duration and efficacy of drugs in the body.
N-glycoside, a type of oligosaccharide, is a basic core composed of two N-acetylglucosamine and three mannos, and based on this core, several types of monosaccharides are bound, Class. The high mannose N-glycoside is a structure in which only mannose is added to the core, and the complex N-glycoside is a monosaccharide other than mannose, that is, N-acetyl hexosamine, Galactose and sialic acid are bonded to each other. The hybrid type refers to a structure in which monosaccharide corresponding to the complex type is attached to mannose having one side centered on the core.
For the N-linked oligosaccharide analysis, a two-step pretreatment process is carried out: a step of separating the oligosaccharide from the protein to separate and separate the oligosaccharide, and a step of purifying the oligosaccharide to extract and concentrate the separated oligosaccharide.
The step of separating the oligosaccharide is the PNGase F enzyme which is attached to the asparagine for the N-glycoside and selectively cleaves it. This enzyme treatment process takes about 16 ~ 18 hours and therefore it takes up the largest part of the time required for sample treatment.
The sugar chain purification process generally uses solid phase extraction (SPE). Solid phase extraction is a method of separating the mixture to be extracted according to its chemical and physical affinity including polarity, hydrophilicity and acidity. In the case of the mixed sugar sample in which the sugar chain is separated, the salt used in the sample or the salt used in the protein denaturation Therefore, the salt and the remaining protein are removed by solid phase extraction method, and only the sugar chain is separated and purified.
In the oligosaccharide assay, the smaller the amount of the test sample, the lower the reproducibility and sensitivity of the assay, and the more likely it is that the disease marker is not accurately displayed. In addition, the enzyme treatment step for separating the oligosaccharide takes 16-18 hours, which is the longest time in preparation of the N-glycan sample. In addition, in the case of the solid phase extraction method performed directly by the experimenter, it takes a long time to increase the number of samples and it is difficult to ensure high reproducibility, and even when a large amount of samples are processed by two or more experimenters, . In addition, the protein denaturation process before the enzymatic treatment of the sample may cause a difference in the experimental method due to the error of the pipetting that may occur during the repeated experiment or the mistake of the experimenter due to the long time experiment in the case of a large number of samples, If the experimenter performs the procedure, the reproducibility may be lost due to errors caused by individual differences. Due to the limitations of the prior art as described above, the construction and optimization of a high-speed large-capacity automated sample pretreatment system is required to analyze the products of various batches, to characterize the oligosaccharide in the biopharmaceutical industry, to identify oligosaccharide markers, There is a continuing need in all aspects.
Ovarian cancer, a typical female disease, is the second most common cancer among female females. 1,200 to 1,400 patients are newly diagnosed with cancer each year, but usually are found in more than three cases. It is difficult to find, and therefore the treatment and prognosis is poor.
Protein-based ovarian cancer biomarkers such as CA125 and CA15-3, which are currently being used in clinical practice, have low specificity and sensitivity with an area under the curve (AUC) of less than 0.6, which makes accurate diagnosis difficult. Such as the need for additional invasive diagnostic methods. However, if invasive methods such as biopsy are used, diagnosis rate can be increased, but it will cause economic and physical burden on the patient, and additional time, manpower, and cost will be required in the clinic. Therefore, a new diagnostic marker for ovarian cancer that can be quickly diagnosed with high diagnostic yield, short time, and low cost is required.
The above-described conventional oligosaccharide sample pretreatment system is capable of solving the problems of sample pretreatment for analyzing the conventional oligosaccharide, enhancing reproducibility with a small amount of sample in a short time and treating a large amount of sample, It is an object of the present invention to detect and provide non-invasive ovarian cancer markers using body fluids such as ascites.
Features of the present invention will now be discussed.
(One) 24 wells Sample Processing Platform
In the 24 well platform of the present invention, galvane profiling and relative quantitative analysis were performed using a sample (25 μl) smaller than the amount (50-100 μl) of the sample currently used for analyzing body fluid glucose, thereby ensuring sufficient reproducibility , And ovarian cancer biomarkers.
The 24 well platform of the present invention shortens the enzyme treatment step, which required 16-18 hours in the past, to around 10 minutes.
The 24 well platform of the present invention uses the same cartridge as the graphitized carbon cartridge used in the manual process using a syringe pump or the conventional process using a vacuum manifold.
In the case of the solid phase extraction method performed directly by the experimenter, it takes a long time to increase the number of samples and it is difficult to ensure high reproducibility, and even when a large amount of samples is treated by two or more experimenters, The whole process of solid phase extraction was automated by using a liquid handler system and the same experimental procedure was applied to all the samples to increase the reproducibility and reduce labor and time required for sample preparation.
(2) 96 wells Sample Processing Platform
We developed a 96-well sample handling platform that increased the number of treated samples while maintaining the fundamental advantages of a 24-well platform and applied it to other types of liquid handler systems.
In the case of a 96-well platform, a new graphitized carbon cartridge plate in the form of a well different from the conventional graphitized carbon cartridge is used due to an increase in the number of samples. Therefore, based on the existing solid phase extraction process, a new protocol was created to fit the 96 well platform and the protein modification process, which was performed manually on the 24 well platform, was automated.
For the 96 well platform, the galvanic profiling was performed using less sample volume (15 μl) to ensure sufficient reproducibility.
According to the conventional method, the protein denaturation process before the enzyme treatment of the sample may cause a difference in the experimental method due to the error of the pipetting which may occur during the repeated experiment or the mistake of the experimenter due to the long time experiment, Although more than one experimenter may lose reproducibility due to errors due to individual differences in the course of the experiment, the automation of the protein denaturation process on the 96 well platform of the present invention is also minimized to reduce the reproducibility that can occur in large samples.
(3) Sugar chain Diagnosis of ovarian cancer detected by simultaneous use of analytical sample preparation system and high sensitivity high resolution mass spectrometer Marker
The present invention
A method for detecting a non-invasive ovarian cancer marker in a body fluid sample such as blood, serum, and ascites by using a high-speed, large-capacity automated N-glycan sample preprocessing system and a high-sensitivity high-
The high-speed, high-capacity automated N-glycan sample pretreatment system comprises: preparing a body fluid sample; Protein denaturation in serum samples; Treating the protein denatured sample with a PNGase F enzyme to cleave the N-glycoside from the protein; Adding ethanol to precipitate the protein and separating the N- glycans of the supernatant; And concentrating and purifying the separated N-oligosaccharide,
In the body fluid sample preparation step, 20 or more samples are listed in a single batch, each sample takes 15 to 30 μl,
In the protein denaturation step, the container containing the sample is alternately put in a liquid of 70 to 90 ° C and a liquid of normal temperature for 10 minutes to 20 seconds, for 1 minute and 30 seconds to 5 minutes, or the container containing the sample is placed in a heating rack, Deg.] C for 1 minute to 2 minutes, cooled to room temperature,
In the PNGase F enzyme treatment step, an enzyme is added to the protein denatured sample, and the enzyme is then subjected to a microwave reactor at 30 to 38 ° C for 5 to 20 minutes.
The separation and purification of the separated N-glycoside is carried out by a solid phase extraction method using a graphitized carbon cartridge using a liquid sample handling automation system, but washing, sample loading, desalting and elution are performed by applying a pressure of 150-400 mbar Lt; RTI ID = 0.0 > non-invasive < / RTI > ovarian cancer.
The present invention relates to a method for producing N-
(Hex) 3 (HexNAc) 2 (Fuc) 1 Theoretical single isotope mass value 1056.386,
(Hex) 3 (HexNAc) 3 Theoretical single isotope mass value 1113.407,
(Hex) 5 (HexNAc) 2 Theoretical Single Isotope Mass Value 1234.433,
(Hex) 4 (HexNAc) 3 Theoretical single isotope mass value 1275.460,
(Hex) 4 (HexNAc) 3 (Fuc) 1 Theoretical single isotope mass value 1421.518,
(Hex) 5 (HexNAc) 3 Theoretical Single Isotope Mass Value 1437.513,
(Hex) 4 (HexNAc) 4 Theoretical single isotope mass value 1478.539,
(Hex) 3 (HexNAc) 5 Theoretical Single Isotope Mass Value 1519.566,
(Hex) 4 (HexNAc) 3 (NeuAc) 1 Theoretical single isotope mass value 1566.555,
(Hex) 5 (HexNAc) 3 (Fuc) 1 Theoretical single isotope mass value 1583.571,
(Hex) 6 (HexNAc) 3 Theoretical Single Isotope Mass Value 1599.566,
(Hex) 4 (HexNAc) 4 (Fuc) 1 Theoretical single isotope mass value 1624.597,
(Hex) 4 (HexNAc) 5 Theoretical Single Isotope Mass Value 1681.619,
(Hex) 5 (HexNAc) 4 (Fuc) 1 Theoretical single isotope mass value 1786.650,
(Hex) 5 (HexNAc) 3 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass value of 1874.666,
(Hex) 9 (HexNAc) 2 Theoretical single isotope mass value of 1882.645,
(Hex) 6 (HexNAc) 3 (NeuAc) 1 Theoretical single isotope mass value 1890.661,
(Hex) 4 (HexNAc) 5 (NeuAc) 1 Theoretical Single Isotope Mass Value 1972.714,
(Hex) 10 (HexNAc) 2 Theoretical single isotope mass value 2044.697,
(Hex) 5 (HexNAc) 4 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass value 2077.745,
(Hex) 6 (HexNAc) 4 (NeuAc) 1 Theoretical single isotope mass value 2093.740,
(Hex) 4 (HexNAc) 5 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass value 2118.772,
(Hex) 6 (HexNAc) 5 (Fuc) 1 Theoretical single isotope mass value 2151.782,
(Hex) 6 (HexNAc) 4 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass value 2239.798,
(Hex) 5 (HexNAc) 5 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass value 2280.825,
(Hex) 7 (HexNAc) 6 (NeuAc) 1 Theoretical single isotope mass values of 2661.952 and
(Hex) 7 (HexNAc) 6 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass value of at least 2808.010,
The present invention relates to a method for detecting non-invasive ovarian cancer in a body fluid sample characterized by the fact that when the mass spectrometry shows a frequency of 80% or more in the whole sample and the relative content is expressed by a difference of more than 10% (However, HexNAc, N-acetylhexosamine; Hex, Hexose; Fuc, Fucose; NeuAc, N-acetylneuraminic acid}.
In addition,
The separated, concentrated and purified N-
(Hex) 5 (HexNAc) 3 (Fuc) 1 Theoretical single isotope mass value 1583.571,
(Hex) 6 (HexNAc) 3 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass value 2036.719,
(Hex) 5 (HexNAc) 5 (Fuc) 2 Theoretical single isotope mass value 2135.787,
(Hex) 6 (HexNAc) 5 (Fuc) 1 Theoretical single isotope mass values of 2151.782 and
(Hex) 5 (HexNAc) 5 (Fuc) 1 (NeuAc) 2 theoretical single isotope mass value of 2571.92,
In the mass spectrometric assay, when the frequency is more than 10% different from the normal group among the N-oligosaccharide which shows more than 80% of the frequency in the normal group or at least one of the patient group, the ovarian cancer is diagnosed as ovarian cancer ≪ RTI ID = 0.0 > HexNAc, < / RTI >N-acetylhexosamine; Hex, Hexose; Fuc, Fucose; NeuAc, N-acetylneuraminic acid}.
In addition,
Among the oligosaccharides expressed with a relative content of more than 10%
(Hex) 3 (HexNAc) 5 Theoretical Single Isotope Mass Value 1519.566,
(Hex) 4 (HexNAc) 5 Theoretical Single Isotope Mass Value 1681.619,
(Hex) 9 (HexNAc) 2 Theoretical single isotope mass value of 1882.645,
(Hex) 4 (HexNAc) 5 (NeuAc) 1 Theoretical Single Isotope Mass Value 1972.714,
(Hex) 10 (HexNAc) 2 Theoretical single isotope mass value 2044.697,
(Hex) 6 (HexNAc) 4 (NeuAc) 1 Theoretical single isotope mass value 2093.740,
(Hex) 4 (HexNAc) 5 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass values of 2118.772 and
(Hex) 7 (HexNAc) 6 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass value of at least 2808.010,
The present invention relates to a method for analyzing ovarian cancer biomarkers, which can identify the stage of ovarian cancer by increasing the amount according to the progress of ovarian cancer.
In addition, the present invention relates to a method for producing the above-
(Hex) 5 (HexNAc) 2 Theoretical Single Isotope Mass Value 1234.433,
(Hex) 4 (HexNAc) 3 (Fuc) 1 Theoretical single isotope mass value 1421.518,
(Hex) 4 (HexNAc) 4 Theoretical single isotope mass value 1478.539, (Hex) 4 (HexNAc) 4 (Fuc) 1 Theoretical single isotope mass value 1624.597,
(Hex) 5 (HexNAc) 4 (Fuc) 1 Theoretical single isotope mass value 1786.650, (Hex) 5 (HexNAc)
(Hex) 6 (HexNAc) 5 (Fuc) 1 It is at least one of the theoretical single isotope mass values of 2151.782,
And a method for analyzing an ovarian cancer biomarker characterized in that the amount of ovarian cancer is decreased according to the progress of the ovarian cancer, thereby confirming the stage of ovarian cancer.
In addition,
a) preparing a body fluid sample;
b) protein denaturation in the body fluid sample;
c) treating the protein denatured sample with a PNGase F enzyme to cleave the N-glycoside from the protein;
d) N-glycosylation, ethanol is added to precipitate the protein, and the N-glycoside of the supernatant is separated; And
e) analyzing the separated N- glycans by a mass spectrometer,
The N-glycans of step e)
(Hex) 3 (HexNAc) 2 (Fuc) 1 Theoretical single isotope mass value 1056.386,
(Hex) 3 (HexNAc) 3 Theoretical single isotope mass value 1113.407,
(Hex) 5 (HexNAc) 2 Theoretical Single Isotope Mass Value 1234.433,
(Hex) 4 (HexNAc) 3 Theoretical single isotope mass value 1275.460,
(Hex) 4 (HexNAc) 3 (Fuc) 1 Theoretical single isotope mass value 1421.518,
(Hex) 5 (HexNAc) 3 Theoretical Single Isotope Mass Value 1437.513,
(Hex) 4 (HexNAc) 4 Theoretical single isotope mass value 1478.539,
(Hex) 3 (HexNAc) 5 Theoretical Single Isotope Mass Value 1519.566,
(Hex) 4 (HexNAc) 3 (NeuAc) 1 Theoretical single isotope mass value 1566.555,
(Hex) 5 (HexNAc) 3 (Fuc) 1 Theoretical single isotope mass value 1583.571,
(Hex) 6 (HexNAc) 3 Theoretical Single Isotope Mass Value 1599.566,
(Hex) 4 (HexNAc) 4 (Fuc) 1 Theoretical single isotope mass value 1624.597,
(Hex) 4 (HexNAc) 5 Theoretical Single Isotope Mass Value 1681.619,
(Hex) 5 (HexNAc) 4 (Fuc) 1 Theoretical single isotope mass value 1786.650,
(Hex) 5 (HexNAc) 3 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass value of 1874.666,
(Hex) 9 (HexNAc) 2 Theoretical single isotope mass value of 1882.645,
(Hex) 6 (HexNAc) 3 (NeuAc) 1 Theoretical single isotope mass value 1890.661,
(Hex) 4 (HexNAc) 5 (NeuAc) 1 Theoretical Single Isotope Mass Value 1972.714,
(Hex) 10 (HexNAc) 2 Theoretical single isotope mass value 2044.697,
(Hex) 5 (HexNAc) 4 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass value 2077.745,
(Hex) 6 (HexNAc) 4 (NeuAc) 1 Theoretical single isotope mass value 2093.740,
(Hex) 4 (HexNAc) 5 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass value 2118.772,
(Hex) 6 (HexNAc) 5 (Fuc) 1 Theoretical single isotope mass value 2151.782,
(Hex) 6 (HexNAc) 4 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass value 2239.798,
(Hex) 5 (HexNAc) 5 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass value 2280.825,
(Hex) 7 (HexNAc) 6 (NeuAc) 1 Theoretical single isotope mass values of 2661.952 and
(Hex) 7 (HexNAc) 6 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass value of at least 2808.010,
The present invention relates to a non-invasive ovarian cancer biomarker analysis method characterized by the fact that when the mass spectrometry shows a frequency of 80% or more in the whole sample and that the relative amount is expressed by 10% or more in comparison with the normal group , HexNAc, N-acetylhexosamine; Hex, Hexose; Fuc, Fucose; NeuAc, N-acetylneuraminic acid}.
In addition,
a) preparing a body fluid sample;
b) protein denaturation in the body fluid sample;
c) treating the protein denatured sample with a PNGase F enzyme to cleave the N-glycoside from the protein;
d) N-glycosylation, ethanol is added to precipitate the protein, and the N-glycoside of the supernatant is separated; And
e) analyzing the separated N- glycans by a mass spectrometer,
The N-glycans of step e)
(Hex) 5 (HexNAc) 3 (Fuc) 1 Theoretical single isotope mass value 1583.571,
(Hex) 6 (HexNAc) 3 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass value 2036.719,
(Hex) 5 (HexNAc) 5 (Fuc) 2 Theoretical single isotope mass value 2135.787,
(Hex) 6 (HexNAc) 5 (Fuc) 1 Theoretical single isotope mass values of 2151.782 and
(Hex) 5 (HexNAc) 5 (Fuc) 1 (NeuAc) 2 theoretical single isotope mass value of 2571.92,
In a mass spectrometric assay, a non-invasive ovarian cancer characterized by diagnosis of ovarian cancer when the frequency of the N-oligosaccharide that expresses more than 80% of the normal group or the patient group is more than 10% Cancer biomarker assay method (HexNAc, N-acetylhexosamine; Hex, Hexose; Fuc, Fucose; NeuAc, N-acetylneuraminic acid}.
The present inventors have found that an abundance marker for diagnosing ovarian cancer comprising N-
(Hex) 3 (HexNAc) 2 (Fuc) 1 Theoretical single isotope mass value 1056.386,
(Hex) 3 (HexNAc) 3 Theoretical single isotope mass value 1113.407,
(Hex) 5 (HexNAc) 2 Theoretical Single Isotope Mass Value 1234.433,
(Hex) 4 (HexNAc) 3 Theoretical single isotope mass value 1275.460,
(Hex) 4 (HexNAc) 3 (Fuc) 1 Theoretical single isotope mass value 1421.518,
(Hex) 5 (HexNAc) 3 Theoretical Single Isotope Mass Value 1437.513,
(Hex) 4 (HexNAc) 4 Theoretical single isotope mass value 1478.539,
(Hex) 3 (HexNAc) 5 Theoretical Single Isotope Mass Value 1519.566,
(Hex) 4 (HexNAc) 3 (NeuAc) 1 Theoretical single isotope mass value 1566.555,
(Hex) 5 (HexNAc) 3 (Fuc) 1 Theoretical single isotope mass value 1583.571,
(Hex) 6 (HexNAc) 3 Theoretical Single Isotope Mass Value 1599.566,
(Hex) 4 (HexNAc) 4 (Fuc) 1 Theoretical single isotope mass value 1624.597,
(Hex) 4 (HexNAc) 5 Theoretical Single Isotope Mass Value 1681.619,
(Hex) 5 (HexNAc) 4 (Fuc) 1 Theoretical single isotope mass value 1786.650,
(Hex) 5 (HexNAc) 3 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass value of 1874.666,
(Hex) 9 (HexNAc) 2 Theoretical single isotope mass value of 1882.645,
(Hex) 6 (HexNAc) 3 (NeuAc) 1 Theoretical single isotope mass value 1890.661,
(Hex) 4 (HexNAc) 5 (NeuAc) 1 Theoretical Single Isotope Mass Value 1972.714,
(Hex) 10 (HexNAc) 2 Theoretical single isotope mass value 2044.697,
(Hex) 5 (HexNAc) 4 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass value 2077.745,
(Hex) 6 (HexNAc) 4 (NeuAc) 1 Theoretical single isotope mass value 2093.740,
(Hex) 4 (HexNAc) 5 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass value 2118.772,
(Hex) 6 (HexNAc) 5 (Fuc) 1 Theoretical single isotope mass value 2151.782,
(Hex) 6 (HexNAc) 4 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass value 2239.798,
(Hex) 5 (HexNAc) 5 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass value 2280.825,
(Hex) 7 (HexNAc) 6 (NeuAc) 1 Theoretical single isotope mass values of 2661.952 and
(Hex) 7 (HexNAc) 6 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass value of 2808.010 was detected,
Also, as a frequency marker for ovarian cancer diagnosis including N-
(Hex) 5 (HexNAc) 3 (Fuc) 1 Theoretical single isotope mass value 1583.571,
(Hex) 6 (HexNAc) 3 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass value 2036.719,
(Hex) 5 (HexNAc) 5 (Fuc) 2 Theoretical single isotope mass value 2135.787,
(Hex) 6 (HexNAc) 5 (Fuc) 1 Theoretical single isotope mass values of 2151.782 and
(Hex) 5 (HexNAc) 5 (Fuc) 1 (NeuAc) 2 theoretical single isotope mass value of 2571.92.
The present inventors used the high-throughput automated oligosaccharide sample pretreatment system and the high-sensitivity high-resolution mass spectrometer simultaneously to discover N-glycan markers of ovarian cancer associated with oligosaccharide mutation from a large amount of samples.
Using the high-throughput automated oligosaccharide sample preparation system of the present invention, samples can be pretreated and assayed in a short time in a large amount of sample. When a high-sensitivity high-resolution mass spectrometer is used, a high diagnostic rate can be expected compared with an existing ovarian cancer biomarker using a protein Markers can be uncovered.
1 is a diagram showing a procedure of analyzing a human body fluid-derived oligosaccharide by the 24-well sample processing system of the present invention.
FIG. 2 is a comparison chart comparing the amount of sample, the time required for each step, the processable capacity, and the pros and cons of the conventional 24-well processing system and the 96-well processing system of the present invention.
Figure 3 shows the arrangement of all samples treated with a 24 well sample processing system in the system of the present invention. 144 patients with ovarian cancer, 202 normal patients, 134 commercial standard samples, total 480 samples.
FIG. 4 is a graph showing the coefficient of variation (CV) for each batch in order to confirm the reproducibility between batches in the 24-well sample processing system of the present invention. The closer the coefficient of variation is to zero, the higher the reproducibility. All four batches showed excellent reproducibility of less than 0.2.
FIG. 5 shows the reproducibility of the 24-well sample processing system of the present invention according to positions in the batch, and reproducibility was confirmed for four batches composed only of commercially available standard samples. The number of graphs represents the corelation coefficient. The closer the correlation coefficient is to 1, the higher the reproducibility.
FIG. 6 is a graph showing the reproducibility of the apparatus used in the oligosaccharide analysis experiment for detecting ovarian cancer biomarkers. The total ion chromatograms were superimposed on all 24 samples of batch No. 13 consisting of commercial standard samples and showed high reproducibility in the oligosaccharide detection zone.
FIG. 7 is a diagram showing a biological synthesis process of the oligosaccharide against the ovarian cancer biomarker list in Table 1. FIG.
8 is a stage marker showing a difference according to the progression stage (stage, stage) of the ovarian cancer among the quantitative markers showing a difference in the relative amount between the patient and the normal person. As the cancer progresses, It is oligosaccharide.
9 is a stage marker, which is a negative marker marker oligosaccharide whose relative content decreases as the cancer progresses.
10 is a diagram showing a procedure for analyzing human serum-derived oligosaccharides by the 96-well sample processing system of the present invention.
11 is a graph showing the reproducibility of the 96-well sample processing system of the present invention. The maximum value and the minimum value of 96 commercial standard samples were 1 and 0.960, respectively.
12 shows files and examples constituting the 96-well sample processing system of the present invention. The entire protocol consists of four files, each of which contains a detailed implementation of the equipment, as shown in the following figure.
FIG. 13 shows the first configuration and proceeding order of the 96-well sample processing system, and the
FIG. 14 is a second configuration and procedure sequence of the 96-well sample processing system, and the file No. 04 in FIG. 12 is implemented in this configuration. The specific pressure or amount of solution may vary depending on the experiment.
Hereinafter, the configuration of the present invention will be described in more detail with reference to specific embodiments. However, it is apparent to those skilled in the art that the scope of the present invention is not limited to the description of the embodiments.
Experimental Method
In the present invention, experiments were conducted using two different liquid handling systems.
(1) 24-well sample processing system (FIG. 1)
A total of 480 serum samples were used in this study: 144 patients with ovarian cancer (Patient; P), 202 patients with normal control (C), and 134 patients with standard (S) standard. Patients and normal subjects were obtained from K hospitals in Seoul. The amount of the sample was the same as 25 μl each.
The three types of samples (P, C, and S) were arbitrarily extracted arbitrarily prior to the experiment to constitute 16 batches, and the same commercial standard samples were added to the middle and each batch of batches (1, 7, 13, 20 Four batches). One batch consisted of 24 samples (6 × 4) and the same batch was sampled at one time.
For the separation of smooth N-glycoside, the serum sample first removed the tertiary structure through the protein denaturation process. 25 μl of serum samples were mixed with 25 μl of 200 mM NH 4 HCO 3 (containing 10 mM dithiothreitol), vigorously stirred, and then treated with hot water and cold water at 90 ° C for 10 seconds alternately for 2 minutes to induce protein denaturation, To prevent protein aggregation.
Samples were treated with PNGase F (peptide N-glycosidase F; 500,000 units / ml) purchased from New England BioLabs (Ipswich, Mass.) To extract N- glycans from the samples. 1 μl (500 units) of PNGase F was added to the protein-denatured sample and cultured in a microwave reactor (400 W, 37 ° C, 10 min) to separate N- glycans.
After 200 μl of cold ethanol was added to the sample after completion of the N-glycosidase cleavage reaction, the sample was allowed to stand at -45 ° C. for one hour to separate the N-glycoside and precipitate the remaining protein.
After protein precipitation, centrifugation was carried out at 4 ° C for 20 minutes at 14,400 rpm, and then 200 μl of supernatant was taken for each sample to completely dry.
1 ml of ultrapure water was added to each dried sample and stirred vigorously to prepare a sugar chain sample for separation and purification.
Solid phase extraction (SPE) using a graphitized carbon cartridge was applied to separate and purify only N-glycoside from the sample. The solid phase extraction step was carried out using a Biomek FXp Laboratory Automation Workstation, a liquid sample handling system from Beckman coulter. Graphitized carbon cartridges were purchased from Grace. First, the cartridge was washed with 2 ml of ultrapure water, 2 ml of 80% (v / v) acetonitrile (containing 0.1% trifluoroacetic acid) and 2 ml of ultrapure water in this order. Thereafter, a sample of the sugar chain was injected into the graphitized carbon cartridge, and then 4 ml of ultrapure water was poured to remove the salt. The remaining glycans in the cartridge were eluted sequentially with 2 ml of 20% (v / v) acetonitrile, 40% (v / v) acetonitrile (containing 0.05% trifluoroacetic acid). Each fraction fraction was collected, dried with a vacuum centrifugal evaporator and dissolved in 15 μl of ultrapure water prior to mass spectrometry.
Mass-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry (Ulterflextreme, Bruker daltonics) was used for mass spectrometry to confirm reproducibility of the invention. As the matrix, 2,5-dihydroxybenzoic acid (2,5-DHB) was used. The 20% acetonitrile fraction was applied to the plate in the order of 1 μl of sample, 0.3 μl of 0.01 M NaCl (dopant) and 0.5 μl of matrix, followed by vacuum drying and analysis in positive mode. The 40% acetonitrile fraction was applied to the plate in the order of 1 μl of the sample and 0.8 μl of the matrix, followed by vacuum drying and analysis in negative mode.
Mass spectrometry for biomarker extraction was performed using a nanoLC chip / Q-TOF mass spectrometer (6530 Q-TOF, Agilent technologies). The chips used were porous graphitized carbon chips from Agilent, Inc., which are the same as those used in the apparatus. The mobile phase was (A) 3% acetonitrile (containing 0.1% formic acid) and (B) 90% acetonitrile (containing 0.1% formic acid), with a total assay time of 50 minutes per sample.
The results of MALDI-TOF MS were analyzed by using FlexAnalysis (version 3.3 build 75, Bruker) and the peak list was extracted with S /
(2) 96-well sample processing system (FIG. 10)
The large volume sample automation platform established in the above experiment was applied to other types of liquid sample handling systems. Reproducibility tests were carried out using 96 commercial standard samples. The amount of sample was 15 μl, which is smaller than that of the 24 - well sample processing system. 96 samples were in one batch (12 x 8) and samples in the same batch were processed at once using a 96 well PCR plate.
All processes except the N-glycosylation process and the protein precipitation process using microwave reactor were automated, and the automated process was performed using Agilent's Encore Multispan Liquid Handling System.
The tertiary structure of the serum sample was removed through protein denaturation. 15 μl of the serum sample was mixed with 15 μl of 200 mM NH 4 HCO 3 (containing 10 mM dithiothreitol), mixed in a shaker, and placed in a heating rack at 80 ° C. for 1 minute to denature the protein.
After the protein denatured sample was cooled for 2 minutes at room temperature, 5 μl of a mixed solution of 1 μl of PNGase F and 4 μl of 100 mM NH 4 HCO 3 (containing 5 mM dithiothreitol) was added, and the mixture was transferred to a microwave reactor (400 W, 37 ° C., To separate N- glycans.
After the N-glycosylase cleavage reaction was completed, 140 μl of cold ethanol was added to the sample, and the mixture was allowed to stand at 45 ° C for 1 hour to separate the N-glycoside and precipitate the remaining protein.
After precipitation, centrifugation was carried out at 4 ° C for 40 minutes at 4,680 rpm, and then 140 μl of supernatant was taken for each sample and completely dried.
800 쨉 l of ultrapure water was added to each dried sample, and stirred in a shaker to prepare a sugar chain sample for separation and purification.
Solid phase extraction (SPE) using a graphitized carbon cartridge was applied to separate and purify only N-glycoside from the sample. A 96-well cartridge plate was used and purchased from Thermo Scientific. First, the cartridge was washed with 1 ml of ultrapure water, 1 ml of 80% (v / v) acetonitrile (including 0.1% trifluoroacetic acid) and 1 ml of ultrapure water in this order. Thereafter, a sample of the sugar chain was injected into the cartridge, and 1 ml of ultrapure water was poured to remove the salt. The remaining oligos in cartridges were eluted in the order of 1 mL each of 20% (v / v) acetonitrile, 40% (v / v) acetonitrile (containing 0.05% trifluoroacetic acid). Each fraction fraction was collected and dried with a vacuum centrifugal evaporator and dissolved in 10 μl of ultrapure water prior to mass spectrometry.
The mass spectrometry for confirmation of reproducibility and the processing of MALDI-TOF MS analysis results are the same as those of the 24 sample processing system.
result
The results of the above experiment will be described in detail with reference to the drawings.
1) Fig. 1 is a conceptual diagram of a 24-well sample processing system in a high-speed large capacity automated sugar chain pretreatment system of the present invention.
For the N - glycosylation process using PNGase F, a microwave reactor was used for enzyme reaction. The enzyme treatment process using the conventional heating bath takes 16-18 hours, whereas the enzyme treatment process using the microwave reactor in the present invention takes 10 minutes and the entire time can be shortened.
The extraction and purification process of N-glycoside using solid phase extraction method used an automated liquid sample handling system.
2) FIG. 2 is a table summarizing the differences between the conventional serum sample pretreatment method and the present invention method.
In the present invention, a small amount of sample of 25 μl for 24 wells and 15 μl for 96 wells was used.
Further, in the present invention, the enzyme reaction time for separating N-glycosylase was shortened from 16 to 18 hours to 10 minutes.
Further, in the present invention, 24 or 96 samples can be treated at a time in the extraction and purification step.
In addition, the present invention ensures high reproducibility by the same experimental method using an automated system.
3) The upper part of FIG. 3 is brief information on serum samples of ovarian cancer patients used in the examples of the present invention. Ovarian cancer is classified according to progression stage, and "R" represents recurrence, ie, recurrence. When the progress step is unknown, it is indicated as "Unknown". "Median Age" refers to the median age of patients, and "Mean Age (SD)" refers to mean age (standard deviation).
The bottom of Fig. 3 is the arrangement of the entire sample treated by the method of the present invention. 144 patients with ovarian cancer, 202 normal patients, 134 commercial standard samples, total 480 samples.
Four batches of
Sixteen batches of batches 2-6, 8-12, and 14-19 consisted of patient, normal, and commercial standard samples and were appropriately randomized.
4) Fig. 4 confirms the reproducibility of the four batches composed of commercial standard samples by confirming the inter-batch reproducibility of the present invention. As a result, the Coefficient of variation (CV) value was 0.2 or less, confirming the inter-batch reproducibility.
5) FIG. 5 confirms the reproducibility of each position in a batch for four batches constituted only by commercial standard samples by confirming the reproducibility of each position in the batch of the present invention. Reproducibility was confirmed by correlation coefficient values based on the frequency and intensity of the detected N-glycans. In this case, the closer the correlation coefficient is to 1, the higher the reproducibility.
For
For
For
For
In the left figure, the yellow circle shows the difference in the reproducibility with the samples in other positions. In the four positions, the one position does not appear constantly and irregularly. Even if there was a difference in reproducibility, the maximum difference was only 4.6%.
6) Figure 6 shows the reproducibility of the apparatus used in the ovarian cancer biomarker experiment. The total ion chromatograms of all 24 samples of
7) Tables 1 and 2 are lists of human ovarian cancer biomarkers identified with the system of the present invention. Table 1 is a list of abundance markers obtained by relative quantitation obtained by liquid chromatography-mass spectrometry. The oligosaccharides whose relative contents differ by more than 10% in the oligosaccharides having a frequency of 90% or more in all samples are designated as quantitative markers Respectively. Table 2 lists the frequency markers of human ovarian cancer frequencies, and the frequency markers were selected for oligosaccharides with a frequency of more than 80% and a difference of more than 10% between the patient and one of the two groups.
The "Composition" in each table represents the composition of the N-glycoside, and the four numbers are, in order, N-acetylhexosamine, Fucose, N-acetylneuraminic acid. For example, "5410" means an N-glycoside consisting of five hexose residues, four N-acetylhexasamine, and one fucose.
"Rel.abun" in the table means relative content.
"C" and "N" in the table mean "cancer patient" and "normal person" respectively.
The "Class" in the table indicates the type of oligosaccharide. It is abbreviated as HM (high mannose), C (complex), H (hybrid) or CH (complex or hybrid) Indicates which of the hybrids are further attached. That is, F (fucose), S (sialic acid), FS (fucose and siailc acid). "C" is a complex type, "CS" is a complex type and sialic acid is combined, "C-FS" is a hybrid type fucose, and " "H-FS" refers to a hybrid type with fucose and sialic acid combined, and "HF" refers to a hybrid type with fucose bound to a sialic acid. .
8) Table 3 and Table 4 are the tables based on the increase / decrease according to the patient sample for the ovarian cancer biomarker list of Table 1 and Table 2. [ Positive markers are oligosaccharides that increase or increase in frequency in patients and negative markers are oligosaccharides that decrease or decrease in frequency in patients. Most of the lists are quantitative markers, * in the composition is the frequency marker, ** is the quantitative marker and the frequency marker is the oligosaccharide.
9) Fig. 7 is a diagram showing a biological synthesis process of N-glycosylation on the ovarian cancer biomarker list of Table 1 and Table 2. Fig. All oligos are synthesized starting from a precursor having a structure of "10200" and having a different structure after the oligosaccharide is decomposed to the core structure indicated as "3200". White arrows indicate the process by which the oligosaccharide is decomposed from the precursor to the core structure, and the gray arrows indicate the presence of one of N-acetylhexosamine, Fucose, N-acetylneuraminic acid Which means that the structure is further compounded. N-oligosaccharides indicated by bold lines are positive markers that are increased in patients with ovarian cancer, and N-oligosaccharides indicated by dotted lines are negative markers that decrease in ovarian cancer patients. The oligosaccharides that are marked with an asterisk (*) without a border on the oligosaccharide are frequency markers, and the oligosaccharides with a border and an * mark are oligosaccharides that are both quantitative and frequency markers. Sugar-free oligosaccharides are oligosaccharides present in the synthesis process, or oligosaccharides that do not show a significant difference between the normal and the patient.
The positive markers generally contain N-acetylneuraminic acid (8 types out of 12), and even those without N-acetylneuraminic acid start from "3500" and "5512" or "7611" , Which is associated with the biosynthetic process. "10200" and "9200" are precursors and are classified separately in the synthesis process.
Speech markers are mostly composed of fucose structures (11 types out of 18), and even if the structure does not contain fucose, the structure is such that sugar chains are decomposed and synthesized one by one from "5200" to "6510" . The structure extending from "6311" to "6411" contains N-acetylneuraminic acid, but has a fucose structure in the final synthetic sugar chain and exhibits the characteristics of a hybrid type structure.
10). FIG. 8 is a stage marker showing a difference depending on the progress of ovarian cancer among the quantitative markers showing a difference in the relative content between the patient and the normal person, and is a positive cancer marker oligomer in which the relative content increases as the cancer progresses. The amount of marker oligosaccharide increases with progression to the first stage,
11) FIG. 9 is a stage marker, which is a negative marker marker oligosaccharide whose relative content decreases as the cancer progresses. In the normal group, the amount is the highest, and the amount of the marker oligos decreases as the cancer progresses to the first stage, the second stage, the third stage, and the fourth stage.
12) Fig. 10 is an overall conceptual diagram of a 96 well platform in a high-throughput automated oligosaccharide sample preprocessing system. The 96-well sample pretreatment system automated all procedures except for the incubation step after the enzymatic treatment to separate the N-glycoside and the protein precipitation with ethanol using a liquid sample handling system. The N-glycoside separation process using PNGase F was performed using a microwave reactor.
13) Figure 11 shows the reproducibility of the 96-well platform. Reproducibility was confirmed by correlation coefficient values based on the frequency and intensity of the detected N-glycans. In this case, the closer to 1, the higher the reproducibility. The maximum value and the minimum value of 96 commercial standard samples were 1 and 0.960, respectively.
14) FIG. 12 shows files and embodiments constituting the 96-well sample processing system of the present invention. The entire protocol consists of four files, each of which contains a detailed implementation of the equipment, as shown in the following figure.
15) FIG. 13 shows the first configuration and procedure of the 96-well sample processing system, and the
01. Protein denaturation
Before starting the protocol, set the temperature of the heating rack (3) at 80 ℃.
1. Buffer transfer
Start with the sample (15 μl) in a 96-well PCR plate (1) ②.
(2)
(3) ② at 800 rpm for 30 seconds.
2. Protein denaturation
(1) Transfer the PCR plate (2) to the heating rack (3) and heat it for 1 minute.
(2) Move the PCR plate to
3. With PNGase F
(1) Transfer 5 μl of a mixed solution of 1 μl of PNGase F contained in (1) and 4 μl of 100 mM NH 4 HCO 3 (containing 5 mM dithiothreitol) to ④.
(2) Move the
※ Agitated PCR The plates were transferred to a microwave reactor Sugar chain Separation reaction is carried out . After implementation PCR Move plate to ⑤.
02. Ethanol precipitation (migration)
1. Ethanol transfer
(1) Remove 140 ㎕ of cold 100% ethanol in ⑥ to ⑤.
※ Leave at -45 ° C for 1 hour, centrifuge after protein precipitation.
03. Ethanol precipitation ( Supernatant )
1. Upper collection
(1) Transfer the supernatant from step (5) to step (7) in 140 μl increments.
※ The supernatant 12 Workspace Move to ② of ②.
The steps shown above are performed without using the automation system.
16) FIG. 14 is a second configuration and proceeding sequence of the 96-well sample processing system, and the file No. 04 in FIG. 12 is performed in this configuration. The specific pressure or amount of solution may vary depending on the experiment.
04. Solid phase extraction
1. Dry sample dissolution (Reconstitution)
(1) Transfer ultrapure water contained in (1) into a deep well plate containing dried sample (2) in an amount of 800 μl, and stir at 1000 rpm for 1 minute.
(2) Move the plate to
2. Cartridge Cleaning (Conditioning)
(1)
(2)
(3)
3. Sample loading
(1) Pour the sample at the position of ③ into the cartridge of ④ and lower it at a pressure of 200 mbar for 30 seconds.
(2) The plate of ③ moves back to ②.
4. Salt Removal
(1)
5. 20% elution
(1) Move the eluting plate of ⑧ to the inside of vacuum chamber under ④.
(2)
(3) Move the elution plate to
6. 40% elution
(1) Move the
(2)
(3) Move the elution plate back to
7. Transfer of 20% eluate
(1) Move the sample from the 20% elution plate of ③ to the 24 well x 4 tube of ⑩.
(2) The empty elution plate moves to (8).
8. Transfer of 40% eluate
(1) Move the 40% elution plate of ⑨ to ③.
(2) Move the sample from the 40% elution plate of ③ to the 24 well x 4 tube of ⑪.
In the present invention, the amount of the sample was first adjusted in order to prepare a bulk sugar sample for mass-sugar chain analysis. A solid extraction cartridge in the form of a plate different from the conventional method is used, and the amount of elution is changed according to the capacity of the cartridge, so that adjustment of the initial sample amount is inevitable.
When 25 μl and 15 μl of the sample were confirmed, it was confirmed that there was no difference in the results of 100 μl and 50 μl of the conventional methods and in the total number of oligosaccharide profiling and oligosaccharide.
In the present invention, since the protein denaturation step is performed using the liquid sample handling system, the denaturation time and temperature are optimized (80 DEG C, 1 minute).
In addition, since the amount of each sample was reduced in the present invention, the amount of buffer solution and the like were adjusted accordingly in all subsequent steps. The amount of washing, desalting, elution, and elution fraction were controlled.
In addition, the present invention optimizes pressure and time when eluting with a pump in a solid extraction cartridge.
In addition, in the present invention, reproducibility between different batches or positions in the same batch is maximized.
In addition, in the present invention, the entire experiment time is shortened by adjusting the pipetting speed, the position of the test tube, and the moving speed.
Claims (7)
The N-oligosaccharide is found in more than 80% of the total samples in normal and patient groups
(Hex) 3 (HexNAc) 2 (Fuc) 1 Theoretical single isotope mass value 1056.386,
(Hex) 3 (HexNAc) 3 Theoretical single isotope mass value 1113.407,
(Hex) 5 (HexNAc) 2 Theoretical Single Isotope Mass Value 1234.433,
(Hex) 4 (HexNAc) 3 (Fuc) 1 Theoretical single isotope mass value 1421.518,
(Hex) 5 (HexNAc) 3 Theoretical Single Isotope Mass Value 1437.513,
(Hex) 4 (HexNAc) 4 Theoretical single isotope mass value 1478.539,
(Hex) 4 (HexNAc) 3 (NeuAc) 1 Theoretical single isotope mass value 1566.555,
(Hex) 5 (HexNAc) 3 (Fuc) 1 Theoretical single isotope mass value 1583.571,
(Hex) 6 (HexNAc) 3 Theoretical Single Isotope Mass Value 1599.566,
(Hex) 4 (HexNAc) 4 (Fuc) 1 Theoretical single isotope mass value 1624.597,
(Hex) 5 (HexNAc) 3 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass value of 1874.666,
(Hex) 9 (HexNAc) 2 Theoretical single isotope mass value of 1882.645,
(Hex) 6 (HexNAc) 3 (NeuAc) 1 Theoretical single isotope mass value 1890.661,
(Hex) 4 (HexNAc) 5 (NeuAc) 1 Theoretical Single Isotope Mass Value 1972.714,
(Hex) 10 (HexNAc) 2 Theoretical single isotope mass value 2044.697,
(Hex) 5 (HexNAc) 4 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass value 2077.745,
(Hex) 6 (HexNAc) 4 (NeuAc) 1 Theoretical single isotope mass value 2093.740,
(Hex) 4 (HexNAc) 5 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass value 2118.772,
(Hex) 6 (HexNAc) 5 (Fuc) 1 Theoretical single isotope mass value 2151.782,
(Hex) 6 (HexNAc) 4 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass value 2239.798,
(Hex) 5 (HexNAc) 5 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass value 2280.825,
(Hex) 7 (HexNAc) 6 (NeuAc) 1 Theoretical single isotope mass values of 2661.952 and
(Hex) 7 (HexNAc) 6 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass value of at least 2808.010,
The present invention relates to a method for diagnosing ovarian cancer, which comprises diagnosing ovarian cancer when the relative amount of the N-oligosaccharide in the patient group is 10% higher or lower than that in the normal group, using non-invasive ovarian cancer using an abundance marker How to analyze {However, HexNAc, N-acetylhexosamine; Hex, Hexose; Fuc, Fucose; NeuAc, N-acetylneuraminic acid}.
The N-oligosaccharide is found in more than 80% of the total samples in normal and patient groups
(Hex) 5 (HexNAc) 3 (Fuc) 1 Theoretical single isotope mass value 1583.571,
(Hex) 6 (HexNAc) 3 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass value 2036.719,
(Hex) 5 (HexNAc) 5 (Fuc) 2 Theoretical single isotope mass value 2135.787,
(Hex) 6 (HexNAc) 5 (Fuc) 1 Theoretical single isotope mass values of 2151.782 and
(Hex) 5 (HexNAc) 5 (Fuc) 1 (NeuAc) 2 Theoretical single isotope mass value of at least 2571.92,
Wherein the ovarian cancer is diagnosed as ovarian cancer when the frequency of the at least one N-type oligosaccharide is higher or lower than 10% in a patient group sample as compared with the normal group. In a body fluid sample, which is characterized by a non-invasive ovarian Methods for the analysis of cancer {however, HexNAc, N-acetylhexosamine; Hex, Hexose; Fuc, Fucose; NeuAc, N-acetylneuraminic acid}
The N-oligosaccharide in which the relative content of the patient group sample is expressed by 10% higher or lower than that of the normal group
(Hex) 9 (HexNAc) 2 Theoretical single isotope mass value of 1882.645,
(Hex) 4 (HexNAc) 5 (NeuAc) 1 Theoretical Single Isotope Mass Value 1972.714,
(Hex) 10 (HexNAc) 2 Theoretical single isotope mass value 2044.697,
(Hex) 6 (HexNAc) 4 (NeuAc) 1 Theoretical single isotope mass value 2093.740,
(Hex) 4 (HexNAc) 5 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass values of 2118.772 and
(Hex) 7 (HexNAc) 6 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass value of at least 2808.010,
A method for analyzing non-invasive ovarian cancer using an abundance marker in a body fluids sample characterized in that the amount of the N-glycosylation is increased in the sample group according to the progress of the ovarian cancer, .
The N-oligosaccharide in which the relative content of the patient group sample is expressed by 10% higher or lower than that of the normal group
(Hex) 5 (HexNAc) 2 Theoretical Single Isotope Mass Value 1234.433,
(Hex) 4 (HexNAc) 3 (Fuc) 1 Theoretical single isotope mass value 1421.518,
(Hex) 4 (HexNAc) 4 Theoretical single isotope mass value 1478.539,
(Hex) 4 (HexNAc) 4 (Fuc) 1 Theoretical single isotope mass value 1624.597,
(Hex) 5 (HexNAc) 4 (Fuc) 1 (NeuAc) 1 Theoretical single isotope mass values of 2077.745 and
(Hex) 6 (HexNAc) 5 (Fuc) 1 It is at least one of the theoretical single isotope mass values of 2151.782,
A method for analyzing non-invasive ovarian cancer using a abundance marker in a body fluid sample characterized in that the amount of the N-glycoside in the patient sample is decreased according to the progress of the ovarian cancer, .
In the body fluid sample preparation step, 20 or more samples are listed in a single batch, each sample takes 15 to 30 μl,
In the protein denaturation step, the container containing the sample is alternately put in a liquid of 70 to 90 ° C and a liquid of normal temperature for 10 minutes to 20 seconds, for 1 minute and 30 seconds to 5 minutes, or the container containing the sample is placed in a heating rack, Deg.] C for 1 minute to 2 minutes, cooled to room temperature,
In the N-chain cleavage step, PNGase F enzyme is added to the protein denatured sample, and the resulting mixture is subjected to a microwave reactor at 30 to 38 ° C for 5 to 20 minutes.
The separated N-sugar chain is concentrated and purified by a solid phase extraction method using a graphitized carbon cartridge using a liquid sample handling automation system, but washing, sample loading, desalting and elution are carried out by applying a pressure of 150-400 mbar Methods for analyzing non-invasive ovarian cancer.
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