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Glycoprotein group quantitating method by lectin enriching and <18>O marking combined custom algorithm
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A glycoprotein, 18O technology, applied in the biological field, can solve the problems of insufficient data processing power and inability to determine the change of sugar chain structure, and achieve the effect of maintaining accuracy
Inactive Publication Date: 2014-07-09
ZHONGSHAN HOSPITAL FUDAN UNIV
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But this 3 18 O’s marking technology does not have mature computer automatic processing algorithms and special software, so it can only rely on manual calculations, which is also a deficiency pointed out by previous developers (Liu et a1. Proteome Res 9, 227-236, 2010) , the data processing of complex biological samples with a large sample size is obviously insufficient, and this method is still unable to determine the specific sugar chain structure changes in glycoproteins that undergo glycosylation changes
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Embodiment 1
[0051] Embodiment 1: establishment of methodology
[0052] Such as figure 1 Shown, the concrete steps of glycoprotein quantitative method of the present invention are:
[0053] The first step: standard glycoprotein sample preparation:
[0054] Standard glycoprotein bovine fetuin (Bovine fetuin, Sigma company) 200ug and yeast invertase (Yeast invertase, Sigma company) 200ug, respectively with 50mM NH 4 HCO 3 After dissolution, a protein sample with a concentration of 1ug / uL was obtained;
[0055] The second step: enzymatic hydrolysis of protein samples:
[0056] The protein sample with a concentration of 1 ug / uL obtained in the first step was placed in a water bath at 100° C. for 10 minutes, and after returning to room temperature, dithiothreitol (Dithiothreitol, Sigma Company) was added. The amount of dithiothreitol added was 10mM (concentration in protein sample solution), incubate at 57°C for 30 minutes, then add iodoacetamide (Iodoacetamide, GE Healthcare company), the...
Embodiment 2
[0086] Example 2: Validation of actual clinical samples
[0087] 1. Sample processing:
[0088] Sera from 3 patients with hepatocellular carcinoma (all from Zhongshan Hospital Affiliated to Fudan University) and 3 healthy control groups were mixed in equal amounts, and served as the patients with hepatocellular carcinoma and the control group. ProteoMiner Protein Enrichment Kit (Bio-Rad Inc. ) to remove high-abundance proteins such as albumin and immunoglobulin G. After measuring the protein concentration, take an equal amount of protein for the next step.
[0089] 2. Enzymatic hydrolysis of protein samples: the operation is the same as in Example 1.
[0090] 3. Lectin affinity chromatography:
[0091] ConA, WGA and LCH lectins were selected for affinity chromatography. Among them, the resin for immobilizing ConA, the resin for immobilizing WGA, the corresponding Binding Buffer and Elution Buffer are all from Glycoprotein Isolation Kit, produced by Pierce Company, the resi...
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Abstract
The invention provides a glycoprotein group quantitating method, which comprises the specific steps of dividing serum samples of patients with different diseases or the same disease at different periods into two groups, removing high-abundant protein, determining protein concentration, and taking samples with the same protein content for desalination and enzymolysis; carrying out lectin affinity chromatography on an obtained enzymolysis product so as to obtain corresponding sub glycoprotein groups, and lyophilizing; re-dissolving the two groups of samples with solutions prepared from H2<18>O and H2<16>O, combinedly solidifying pancreatin to carry out C-end marking, re-dissolving the two group of samples with the solutions prepared from H2<18>O and H2<16>O after lyophilization, and combining PNGase to carry out N glycosylation locus marking and lyophilization; mixing and loading the two groups of samples to carry out liquid chromatography-mass spectrum detection, and establishing a custom algorithm based on Mascot Distiller to carry out quantitative data processing. The method disclosed by the invention has sufficient accuracy and repeatability, can keep linearity within the range of 1:10-10:1, and can accurately reflect the glycoprotein changes of saccharide types of different N-saccharide chain structures in the samples.
Description
technical field [0001] The present invention relates to a quantitative method for glycoproteomics, in particular to a lectin enrichment and 18 The invention discloses a glycoprotein group quantification method combined with a custom algorithm, belonging to the field of biotechnology. Background technique [0002] Protein glycosylation is an important post-translational modification, which has important significance for protein folding, stability and activity. Protein glycosylation and glycoform changes play an important role in pathophysiological processes such as chronic inflammation, immune response, cell recognition, and cellular immune regulation, and participate in the malignant transformation of cells during the occurrence of malignant tumors, suggesting that the surrounding Changes in the microenvironment are closely related to the development and invasion of malignant tumors; many known cancer-related biomarkers (Biomarkers) are glycoproteins, such as alpha-fetoprot...
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