CN113480629A - Terminal sialylated isomeric glycopeptide marker for distinguishing liver cancer from normal person and screening method thereof - Google Patents
Terminal sialylated isomeric glycopeptide marker for distinguishing liver cancer from normal person and screening method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biomedicine, and particularly relates to a terminal sialylated isomerized glycopeptide marker for distinguishing liver cancer from normal people and a screening method thereof. In the invention, under specific chromatographic retention time, parent ions are selected in a mass spectrum collision pool for fragmentation, and then all fragment ions enter an ion mobility pool for separation and analysis, and most preferably, the fragment ions are separated and analyzedFinally, performing mass spectrum detection; by terminal sialic acid alpha 2,6 and alpha 2,3 isomers B3 +Fragment ([ NeuAc alpha 2-3/alpha 2-6Gal beta 1-4 GlcNAc)]+M/z 657.24) comparison of the areas of the distribution of the time of arrival of the mobilities, and comparison of the resulting alpha 2,6 and alpha 2,3 isomers B3 +And analyzing the fragment relative ratio to determine 6 candidate terminal sialylated isomeric glycopeptide biomarkers with certain distinguishing capacity or combined distinguishing capacity. The invention is helpful to change the current situation of liver cancer diagnosis and has important clinical value for liver cancer diagnosis.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a terminal sialylated isomerized glycopeptide marker for distinguishing liver cancer from normal people and a screening method thereof.
Background
Glycosylation is a complex post-translational modification (PTM) widely present in organisms, and has important effects on the chemical and biological properties of proteins. On human glycoproteins, sialic acid is a family of acidic 9-carbon sugars; sialic acid is linked to galactose (Gal) residues by either an α 2,3 or α 2,6 linkage under the action of sialyltransferase. Sialylation modification of serum proteins is of great importance in biological processes, including immune cell tracking, microbial attachment, coagulation and inflammatory homeostasis, and therefore, the study of sialic acid linked isoforms is of great importance for a better understanding of the role of glycosylation.
Hepatocellular carcinoma is one of the high-grade malignant tumors in China, and the second place of the death rate of the tumor is located. Early diagnosis and early treatment are the key points for improving the survival rate of liver cancer patients. At present, the early diagnosis of liver cancer is mainly carried out clinically by combining Alpha Fetoprotein (AFP) with imaging and pathological examination, but the sensitivity and the specificity are insufficient. With the progress of molecular biological research, new liver cancer markers are continuously discovered, and the early diagnosis of liver cancer is promoted, particularly some glycoproteins and the specific modified sugar chain structures thereof are provided.
Haptoglobin (Hp) is a highly sialylated glycoprotein reported to have aberrant N-glycosylation, such as hepatocellular carcinoma (HCC), in various types of cancer. In previous researches, the fact that the total N-glycan and site-specific sialylated N-glycan on the beta chain of haptoglobin of a liver disease patient are obviously different from those of a healthy person is found, and the fact that the glycan of Hp plays a certain role in the liver cancer generation process is shown. However, N-glycopeptide terminal sialylation ligation isomerism to Hp has not been extensively studied and explored.
Therefore, aiming at the defects of the prior art, the technical personnel in the field provide a class of end sialylated isomeric glycopeptide biomarkers for screening liver cancer or liver disease people, so that early intervention and treatment of the liver cancer are realized, and the mortality of the liver cancer is reduced.
Disclosure of Invention
The invention aims to provide a candidate end sialylated glycopeptide isomerase biomarker for distinguishing liver cancer from normal people and a screening method thereof aiming at the defects of sensitivity and specificity of a marker for early diagnosis of liver cancer so as to change the current situation of diagnosis of liver cancer.
The end sialyl isomerized glycopeptide biomarker provided by the invention is used for distinguishing candidate end sialyl isomerized glycopeptides of liver cancer and normal people, and the number of the end sialyl isomerized glycopeptide biomarker is 6, and the end sialyl isomerized glycopeptide biomarker is respectively as follows:
MVSHH184N#LTTGATLINE-H6N5S1；
MVSHHN#LTTGATLINE-H5N4F1S2；
MVSHHN#LTTGATLINE-H6N5S3；
NLFL207N#HSE-H5N4F1S2；
NLFL207N#HSE-H6N5S3；
VVLHP241N#YSQVD-H6N5F1S3 ；
（HexNAc : N; Hex : H; Fuc : F; NeuAc : S）。
each marker can be distinguished individually, or more in combination.
The invention also provides a screening method of the candidate end sialylated isomerized glycopeptide biomarker of liver cancer and normal people, which comprises the following specific steps:
(1) selecting parent ions of the glycopeptide biomarker in a mass spectrum collision cell for fragmentation under specific chromatographic retention time;
(2) then, all fragment ions enter an ion mobility pool for separation and analysis, and finally mass spectrum detection is carried out;
(3) by terminal sialic acid alpha 2,6 and alpha 2,3 isomers B3 +Fragment ([ NeuAc alpha 2-3/alpha 2-6Gal beta 1-4 GlcNAc)]+M/z 657.24) comparison of the areas of the distribution of the time of arrival of the mobilities, and comparison of the resulting alpha 2,6 and alpha 2,3 isomers B3 +And analyzing the relative fragment ratio to determine the candidate terminal sialylated isomeric glycopeptide biomarker.
The invention provides a screening method of candidate end sialylated isomeric glycopeptide biomarkers of liver cancer and normal people, which comprises the following specific operation processes:
(1) purification of serum haptoglobin: serum samples were centrifuged at 10000-15000 × g for 10-15 minutes and then purified using a HiTrap column. The Hp bound to the column was eluted with elution buffer (pH 3.0, 100 mM Glycine, 0.5M NaCl) and concentrated by acetone precipitation.
(2) Proteolysis of Hp: for serum Hp purified from normal human and liver cancer patients, haptoglobin with a certain volume concentration of 1mg/mL is taken, and the ratio of 1: adding Dithiothreitol (DTT) solution with the concentration of 200-300 mM in the volume ratio of 20, reacting for 0.5-1 h at the temperature of 56-60 ℃, and performing denaturation and reduction treatment on the protein. Then according to the following steps of 1: adding 400-600 mM iodoacetamide solution (IAA) in a volume ratio of 20, and carrying out a light-shielding reaction at room temperature for 0.5-1 h to carry out protein alkylation treatment. Finally, protein and trypsin 50: 1 adding a certain amount of trypsin for enzymolysis of protein, performing enzymolysis at 37 ℃ overnight, adding a certain amount of Glu-C for digestion, and continuing enzymolysis for 12-16 h (according to the ratio of protein: Glu-C = 50: 1). And finally adding 10-20 mu L of formic acid solution to terminate the enzymolysis reaction.
(3) HILIC enriched N-glycopeptide: firstly, using 200-500 μ L of equilibrium liquid (80-75% ACN/0.1% TFA) to balance HILIC column for 3-4 times; after the haptoglobin enzymolysis peptide segment dissolved in the balance liquid is subjected to sample loading for 3-4 times, washing the HILIC column enriched with the glycopeptide for 3-4 times by using the balance liquid; and finally, eluting the N-glycopeptide enriched on the HILIC by 300-500 mu L of 0.1% TFA eluent for 2-4 times, combining the eluates, and carrying out vacuum centrifugal drying for later use.
(4) LC-MS/MS analysis: the online LC adopts an M-Class nano-LC system of Waters company: phase A is 0.1% FA aqueous solution; phase B was 0.1% FA in ACN. The complete N-glycopeptide is subjected to on-column sampling, the sampling flow rate is 0.2-0.3 mu L/min, and the sampling time is 4-5 min; then the mixture enters an analytical column for separation, and a 15-25 cm C18 column (NanoE MZ PST CSH 130C 181.7 mu 75 mu m x 150 mm) is adopted as a chromatographic column: and (3) adopting a gradient of 60-90 minutes, starting from 1-2% of the phase B, starting from 35-40% of the phase B after 40-60 minutes, increasing to 80-85% of the phase B after 5-10 minutes, and then adjusting to 1-2% of the phase B.
(5) N-glycopeptide data retrieval: by adopting ByonicTMSoftware (version 2.16.11, Protein Metrics, San Carlos, Calif.) retrieves the original data file of haptoglobin sequence (P00738), setting the mass errors of parent ions and child ions to 10-20 ppm and 0.03-0.05 Da respectively. The zero deletion cleavage site was digested with trypsin, V8 protease. The fixed modification is carbamoylation (C) and the variable modification is oxidation (M) and deamidation (N). The results were filtered at 1-1.2% FDR and scored by Byonic>A manual verification is performed at a confidence threshold of 150. The retrieved highly reliable N-glycopeptides are used for screening the terminal sialylated isomeric glycopeptide biomarkers in the present invention.
(6) Relative quantitative analysis of N-glycopeptide terminal sialic acid alpha 2,6 and alpha 2,3 connection isomerism in terminal sialylated isomerism glycopeptide biomarker screening: chromatographic conditions consistent with LC-MS/MS were selected. As shown in FIG. 1, at a specific retention time, each complete N-glycopeptide of haptoglobin is selected and subjected to Collision Induced Dissociation (CID) in a quadrupole according to its m/z, all fragments generated by parent ions are subjected to mobility separation in an ion mobility cell (IM), and finally enter a mass spectrometer for detection and signal collection. The spraying voltage is 2.0-2.5 kV, the taper hole voltage is 25-35V, the capillary is heated to 80-120 ℃, and data are acquired in a data-independent mode/data-dependent mode. The mobility traveling wave velocity is 750-1100 m/s, the wave height is set at 35-40V at maximum, N2The flow rate of the drift gas is 90-100 ml/min. The CID energy of the glycopeptide adopts gradient energy from 15V to 50V. The data acquisition primary scanning range is 100-2000 m/z, the resolution is 20K, and the HDMS/MS scanning mode is adopted. Lockspray Mass LE (556.2771 Da) data acquisition software for MassLynx 4.1.
(7) The α 2,6 and α 2,3 isoforms B of each intact N-glycopeptide were analyzed using MassLyn 4.1 software3 +Fragment ([ NeuAc alpha 2-3/alpha 2-6Gal beta 1-4 GlcNAc)]+, m/z 657.24) to extract the mobility Arrival Time Distribution (ATDs); and according to its alpha 2,6 and alpha 2,3 isomers B3The relative proportion of peak areas of the fragments in the distribution time realizes the relative quantitative comprehensive characterization of the complete N-glycopeptide terminal sialic acid alpha 2,6 and alpha 2,3 isomeric connection in serum Hp of normal people and liver cancer patients.
The invention realizes the determination of the candidate end sialyl-isomerism glycopeptide biomarker in serum Hp of normal human and liver cancer patients.
The invention has the beneficial effects that: the invention provides a candidate end sialyl-isomerism glycopeptide biomarker for serum diagnosis of liver cancer. The potential end sialyl isomeric glycopeptide biomarkers can be used independently or jointly, so that the misjudgment defect of a single marker can be avoided, the clinical diagnosis capability can be improved, the current diagnosis situation of liver cancer can be changed, and the potential end sialyl isomeric glycopeptide biomarkers have important clinical values for early discovery and early treatment of the liver cancer.
Drawings
FIG. 1 is a flow chart of the screening and detection of candidate end sialylated isomeric glycopeptide biomarkers for serum Hp of liver cancer.
Fig. 2 is a summary of the results of t-test (two-tailed) for 6 serum Hp candidate terminal sialylated glycopeptide isoforms.
FIG. 3 is a graph of the combined diagnostic ROC for 6 serum Hp candidate terminal sialylated isomeric glycopeptide biomarkers.
Detailed Description
The present invention will be described in further detail with reference to specific examples below so that those skilled in the art can more clearly understand the present invention. The following should not be construed as limiting the scope of the claimed invention.
Example 1
The screening method and the screening process of the candidate terminal sialylated isomeric glycopeptide biomarker contained in the invention are adopted to detect Hp proteins in serum of 27 liver cancer patients and 27 healthy control patients, and finally determine 6 candidate terminal sialylated isomeric glycopeptide biomarkers with certain distinguishing capability or combination distinguishing capability through the relative quantitative comparison and analysis of N-glycopeptide terminal sialic acid linkage isomerism.
The chemicals and solvents used in the examples were all analytical grade.
Reagent required by experiment
(1) Extracting Hp protein in serum: serum samples were centrifuged at 12000 × g for ten minutes and subsequently purified using HiTrap columns.
(2) Sequencing grade trypsin and V8 protease were purchased from Promega (WI, USA).
(II) Experimental procedures
(1) Purification of serum haptoglobin: serum samples were centrifuged at 12000 × g for ten minutes, respectively, followed by purification using HiTrap columns. The Hp bound to the column was eluted with elution buffer (pH 3.0, 100 mM Glycine, 0.5M NaCl) and concentrated by acetone precipitation.
(2) Proteolysis of Hp: for serum Hp purified from normal human and liver cancer patients, 190. mu.l of haptoglobin at a concentration of 1mg/mL was added to 10. mu.l of 200mM Dithiothreitol (DTT) solution, and the mixture was reacted at 56 ℃ for 1 hour to denature and reduce the protein. Then, 10ul of 400mM iodoacetamide solution (IAA) was added thereto, and the mixture was reacted at room temperature for 30 min with exclusion of light to conduct protein alkylation. Finally, 4 mu g of trypsin is added for carrying out enzymolysis on the protein, and after the enzymolysis is carried out for 12 hours at 37 ℃, 4 mu g of Glu-C is added for digestion and the enzymolysis is continued for 12 hours. Finally, 10. mu.L of formic acid solution was added to terminate the enzymatic reaction.
(3) HILIC enriched N-glycopeptide: first, equilibrate HILIC column with 500. mu.L of equilibration solution (80% ACN/0.1% TFA) 3 times; after the haptoglobin enzymolysis peptide segment dissolved in the balance liquid is loaded for 3 times, the HILIC column enriched with glycopeptide is washed for 3 times by the balance liquid; finally, the N-glycopeptide enriched in HILIC was eluted 2 times with 300. mu.L of 0.1% TFA, and the eluates were combined and dried by vacuum centrifugation for further use.
(4) LC-MS/MS analysis: the online LC adopts an M-Class nano-LC system of Waters company: phase A is 0.1% FA aqueous solution; phase B was 0.1% TFA in ACN. Loading the complete N-glycopeptide by using on-column at a flow rate of 0.3 muL/min for 4 min; then the column was separated by a 15 cm C18 column (NanoE MZ PST CSH 130C 181.7. mu.75. mu. m x 150 mm): a gradient of 60 minutes was used, starting from 1% of phase B, starting from 35% of phase B after 40 minutes, increasing to 80% of phase B after 5 minutes, and then adjusting to 1% of phase B.
(5) N-glycopeptide data retrieval: by adopting ByonicTMThe software (version 2.16.11, Protein Metrics, San Carlos, Calif.) searched the raw data file for the haptoglobin sequence (P00738), setting the mass errors of the parent and child ions at 20 ppm and 0.05 Da respectively. The zero deletion cleavage site was digested with trypsin, V8 protease. The fixed modification is carbamoylation (C) and the variable modification is oxidation (M) and deamidation (N). Results were filtered at 1% FDR and Byonic scored>A manual verification is performed at a confidence threshold of 150. The retrieved highly reliable N-glycopeptides are used for screening the terminal sialylated isomeric glycopeptide biomarkers in the present invention.
(6) Relative quantitative analysis of N-glycopeptide terminal sialic acid alpha 2,6 and alpha 2,3 connection isomerism in terminal sialylated isomerism glycopeptide biomarker screening: chromatographic conditions consistent with LC-MS/MS were selected. As shown in FIG. 1, at a specific retention time, each complete N-glycopeptide of haptoglobin is selected and subjected to Collision Induced Dissociation (CID) in a quadrupole according to its m/z, all fragments generated by parent ions are subjected to mobility separation in an ion mobility cell (IM), and finally enter a mass spectrometer for detection and signal collection. The spraying voltage is 2.2 kV, the taper hole voltage is 30V, the heating capillary is 100 ℃, and data are acquired in a non-data-dependent mode/data-dependent mode. The mobility traveling wave velocity is 950 m/s, the wave height is set at 40V, N2The drift gas flow rate was 90 ml/min. The CID energy of the glycopeptide adopts gradient energy from 15V to 50V. The data acquisition primary scanning range is 100-2000 m/z, the resolution is 20K, and the HDMS/MS scanning mode is adopted. Lockspray Mass LE (556.2771 Da) data acquisition software for MassLynx 4.1.
(7) The α 2,6 and α 2,3 isoforms B of each intact N-glycopeptide were analyzed using MassLyn 4.1 software3 +Fragment ([ NeuAc alpha 2-3/alpha 2-6Gal beta 1-4 GlcNAc)]+, m/z 657.24) to extract the mobility Arrival Time Distribution (ATDs); according to alpha 2,6 and alpha 23 isomer B3The relative proportion of peak areas of the fragments in the distribution time realizes the relative quantitative comprehensive characterization of the complete N-glycopeptide terminal sialic acid alpha 2,6 and alpha 2,3 isomeric connection in serum Hp of normal people and liver cancer patients. The invention realizes screening of 42N-glycopeptide sialylation ratios (alpha 2,3 to alpha 2,6 connected with sialic acid) of Hp in serum Hp of normal human and liver cancer patients, and classifies the normal human and liver cell liver cancer patients by adopting orthogonal partial least squares discriminant analysis (OPLS-DA), wherein the VIP value is>The variable of 1 is considered to be related to population differences.
(8) The screening method of the candidate end sialylated isomeric glycopeptide biomarker is adopted to carry out detection and analysis on 6 candidate end sialylated isomeric glycopeptides in Hp protein in serum of 27 liver cancer patients and 27 healthy control patients, and as shown in figure 2, the t test (double-tail) results of the 6 candidate end sialylated isomeric glycopeptides which can distinguish liver cancer from normal people are summarized in 27 normal human serum samples and 27 liver cell liver cancer patient serum samples. FIG. 3 shows the joint ROC plot for 6 candidate terminal sialylated glycopeptides. The 6 candidate terminal sialylated glycopeptides were:
MVSHH184N#LTTGATLINE-H6N5S1；
MVSHHN#LTTGATLINE- H5N4F1S2；
MVSHHN#LTTGATLINE- H6N5S3；
NLFL207N#HSE-H5N4F1S2；
NLFL207N#HSE-H6N5S3；
VVLHP241N#YSQVD-H6N5F1S3。
Claims (3)
1. a terminal sialyl-isomerized glycopeptide biomarker, which is used for distinguishing candidate terminal sialyl-isomerized glycopeptide of liver cancer and normal people; the total number of the medicines is 6, and the medicines are respectively:
MVSHH184N#LTTGATLINE-H6N5S1；
MVSHHN#LTTGATLINE-H5N4F1S2；
MVSHHN#LTTGATLINE-H6N5S3；
NLFL207N#HSE-H5N4F1S2；
NLFL207N#HSE-H6N5S3；
VVLHP241N#YSQVD-H6N5F1S3 ；
each marker is used alone for differentiation, or in combination for differentiation.
2. The method for screening end-sialylated glycopeptide biomarkers of liver cancer and normal human as claimed in claim 1, comprising the steps of:
(1) selecting parent ions of the glycopeptide biomarker in a mass spectrum collision cell for fragmentation under specific chromatographic retention time;
(2) then, all fragment ions enter an ion mobility pool for separation and analysis, and finally mass spectrum detection is carried out;
(3) by terminal sialic acid alpha 2,6 and alpha 2,3 isomers B3 +Comparing the fragment mobility arrival time distribution areas, and comparing the obtained alpha 2,6 and alpha 2,3 isomers B3 +And analyzing the relative fragment ratio to determine the candidate terminal sialylated isomeric glycopeptide biomarker.
3. The screening method according to claim 2, wherein the specific operation flow is as follows:
(1) purification of serum haptoglobin: centrifuging the serum sample at 10000-15000 × g for 10-15 minutes, and then purifying by using a HiTrap column; eluting Hp on the binding column by using an elution buffer solution, and then precipitating and concentrating by using acetone;
(2) proteolysis of Hp: for serum Hp purified from normal human and liver cancer patients, haptoglobin with a certain volume concentration of 1mg/mL is taken, and the ratio of 1: adding a dithiothreitol solution with the concentration of 200-300 mM in a volume ratio of 20, reacting for 0.5-1 h at the temperature of 56-60 ℃, and performing denaturation and reduction treatment on the protein; then according to the following steps of 1: adding 400-600 mM iodoacetamide solution in a volume ratio of 20, and reacting at room temperature in a dark place for 0.5-1 h to carry out protein alkylation; finally, according to the ratio of protein to trypsin 50: 1, adding trypsin for carrying out enzymolysis on the protein, carrying out enzymolysis overnight at 37 ℃, adding Glu-C for digestion, and continuing the enzymolysis for 12-16 h; finally, adding 10-20 mu L of formic acid solution to terminate the enzymolysis reaction;
(3) HILIC enriched N-glycopeptide: firstly, balancing a HILIC column for 3-4 times by using 200-500 mu L of balancing solution; after the haptoglobin enzymolysis peptide segment dissolved in the balance liquid is subjected to sample loading for 3-4 times, washing the HILIC column enriched with the glycopeptide for 3-4 times by using the balance liquid; finally, eluting the N-glycopeptide enriched on HILIC by 300-500 mu L of 0.1% TFA eluent for 2-4 times, combining the eluates, and then carrying out vacuum centrifugal drying for later use;
(4) LC-MS/MS analysis: the online LC adopts an M-Class nano-LC system of Waters company: phase A is 0.1% FA aqueous solution; phase B is 0.1% FA solution in ACN; the complete N-glycopeptide is subjected to on-column sampling, the sampling flow rate is 0.2-0.3 mu L/min, and the sampling time is 4-5 min; and then, separating in an analytical column, wherein the chromatographic column adopts a 15-25 cm C18 column: adopting a gradient of 60-90 minutes, starting from 1-2% of phase B, starting from 35-40% of phase B after 40-60 minutes, increasing to 80-85% of phase B after 5-10 minutes, and then adjusting to 1-2% of phase B;
(5) n-glycopeptide data retrieval: by adopting ByonicTMSearching an original data file of a haptoglobin sequence by software, and setting mass errors of parent ions and child ions to be 10-20 ppm and 0.03-0.05 Da respectively; the zero deletion cleavage site was digested with trypsin, V8 protease; fixed modifications are carbamoylation (C), variable modifications are oxidation (M) and deamidation (N); the results were filtered at 1-1.2% FDR and scored by Byonic>150, performing manual verification under a confidence threshold; the retrieved high credibility N-glycopeptide is used for screening the end sialylation isomeric glycopeptide biomarker;
(6) relative quantitative analysis of N-glycopeptide terminal sialic acid alpha 2,6 and alpha 2,3 connection isomerism in terminal sialylated isomerism glycopeptide biomarker screening: selecting LC-MS/MS consistent chromatographic conditions; under specific retention time, each complete N-glycopeptide of haptoglobin is respectively selected in a quadrupole rod according to m/z of the complete N-glycopeptide and subjected to collision induced dissociation, all fragments generated by parent ions are subjected to mobility separation in an ion mobility pool, and finally the fragments enter a mass spectrum detector for detection and signal collection; the spraying voltage is 2.0-2.5 kV, the taper hole voltage is 25-35V, the capillary is heated to 80-120 ℃, and the data is shownAdopting a non-data-dependent mode/data-dependent acquisition; the mobility traveling wave velocity is 750-1100 m/s, the wave height is set at 35-40V at maximum, N2The flow rate of the drift gas is 90-100 ml/min; gradient energy from 15-50V is adopted for the CID energy of the glycopeptide; the data acquisition primary scanning range is 100-2000 m/z, the resolution is 20K, and the HDMS/MS scanning mode is adopted;
(7) the α 2,6 and α 2,3 isoforms B of each intact N-glycopeptide were analyzed using MassLyn 4.1 software3 +Extracting the mobility arrival time distribution of the fragments; and according to its alpha 2,6 and alpha 2,3 isomers B3The relative ratio of peak areas of the distribution time of the + fragments is used for carrying out relative quantitative comprehensive characterization on the terminal sialic acid alpha 2,6 and alpha 2,3 isomeric connection of the complete N-glycopeptide in serum Hp of a normal human and a liver cancer patient.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060141528A1 (en) * | 2004-05-21 | 2006-06-29 | Aebersold Rudolf H | Compositions and methods for quantification of serum glycoproteins |
| WO2006114659A1 (en) * | 2005-04-26 | 2006-11-02 | Dwek Raymond A | Glycosylation markers for cancer diagnosing and monitoring |
| US20060269974A1 (en) * | 2005-04-26 | 2006-11-30 | Dwek Raymond A | Glycosylation markers for cancer diagnosing and monitoring |
| US20120190576A1 (en) * | 2009-07-14 | 2012-07-26 | Hisashi Narimatsu | Glycan Markers as Measure of Disease State of Hepatic Diseases |
| US20150160232A1 (en) * | 2013-12-09 | 2015-06-11 | Academia Sinica | Automatic amino acid sequencing of glycopeptide by y1 ions |
| US20160289346A1 (en) * | 2013-11-21 | 2016-10-06 | Repetoire Genesis Incorporation | Glycan analysis |
| US20170219590A1 (en) * | 2014-07-22 | 2017-08-03 | National Institute Of Advanced Industrial Science And Technology | Hepatocellular carcinoma marker |
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Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060141528A1 (en) * | 2004-05-21 | 2006-06-29 | Aebersold Rudolf H | Compositions and methods for quantification of serum glycoproteins |
| WO2006114659A1 (en) * | 2005-04-26 | 2006-11-02 | Dwek Raymond A | Glycosylation markers for cancer diagnosing and monitoring |
| US20060269974A1 (en) * | 2005-04-26 | 2006-11-30 | Dwek Raymond A | Glycosylation markers for cancer diagnosing and monitoring |
| US20120190576A1 (en) * | 2009-07-14 | 2012-07-26 | Hisashi Narimatsu | Glycan Markers as Measure of Disease State of Hepatic Diseases |
| US20160289346A1 (en) * | 2013-11-21 | 2016-10-06 | Repetoire Genesis Incorporation | Glycan analysis |
| US20150160232A1 (en) * | 2013-12-09 | 2015-06-11 | Academia Sinica | Automatic amino acid sequencing of glycopeptide by y1 ions |
| US20170219590A1 (en) * | 2014-07-22 | 2017-08-03 | National Institute Of Advanced Industrial Science And Technology | Hepatocellular carcinoma marker |
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