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CN-121988249-A - Sugar chain reduction and ubiquitination device and method

CN121988249ACN 121988249 ACN121988249 ACN 121988249ACN-121988249-A

Abstract

The invention provides a sugar chain reduction and ubiquitination device and method, wherein the device comprises a centrifuge tube and a reaction tube, a tube plug is arranged at the tube orifice of the centrifuge tube, the bottom of the reaction tube penetrates through the tube plug and is inserted into the centrifuge tube, the top and the bottom of the reaction tube are both provided with openings, a supporting layer and an adsorption layer are sequentially arranged in the reaction tube from bottom to top, a reaction cavity of the reaction tube is arranged above the adsorption layer, and the adsorption layer is made of porous graphitized carbon material. The device can realize the integrated operation from the whole process of reducing, desalting and pan-methylation to the secondary desalting of sugar chains, and remarkably reduces the loss caused by transferring samples between different containers.

Inventors

  • WEI JUAN
  • Napier Tuxun Maimaiti
  • GUO SHUHONG

Assignees

  • 上海交通大学

Dates

Publication Date
20260508
Application Date
20260119

Claims (10)

  1. 1. A sugar chain reduction and ubiquitination device is characterized by comprising a centrifuge tube and a reaction tube, wherein a tube plug is arranged at the tube orifice of the centrifuge tube, the bottom of the reaction tube penetrates through the tube plug and is inserted into the centrifuge tube, the top and the bottom of the reaction tube are both provided with openings, a supporting layer and an adsorption layer are sequentially arranged in the reaction tube from bottom to top, a reaction cavity of the reaction tube is arranged above the adsorption layer, and the adsorption layer is made of porous graphitized carbon material.
  2. 2. The sugar chain reduction and ubiquitination device according to claim 1, wherein the support layer is a C8 membrane or a C18 membrane.
  3. 3. The method for producing a sugar chain reduction and ubiquitination device according to claim 1 or 2, characterized by comprising the steps of: S1, filling a supporting layer in a reaction tube, inserting the reaction tube into a centrifuge tube through a tube plug, and fixing the reaction tube in the centrifuge tube through the tube plug; S2, adding a porous graphitized carbon material into a solvent to prepare PGC suspension, adding the PGC suspension into the reaction tube obtained in the step S1, and centrifuging a centrifuge tube to form an adsorption layer; And S3, activating the adsorption layer to obtain the sugar chain reduction and ubiquitination device.
  4. 4. The method for manufacturing a sugar chain reduction and ubiquitination device according to claim 3, wherein S3 is specifically characterized in that acetonitrile solution of trifluoroacetic acid is added into the reaction cavity, a centrifuge tube is centrifuged, acetonitrile aqueous solution containing the trifluoroacetic acid is added into the reaction cavity, the centrifuge tube is centrifuged, aqueous solution of the trifluoroacetic acid is added into the reaction cavity, and the centrifuge tube is centrifuged, so that the sugar chain reduction and ubiquitination device is obtained.
  5. 5. A sugar chain reduction and ubiquitination method, characterized in that the sugar chain reduction and ubiquitination device according to claim 1 or 2 comprises: s1, adding a reducing reagent and an aqueous solution of a sugar chain sample into a reaction cavity to perform a sugar chain reduction reaction; s2, after the reduction reaction is finished, adding water into a reaction cavity of a reaction tube, centrifuging a centrifuge tube, adding water into the reaction cavity, centrifuging the centrifuge tube, and desalting the obtained reducing sugar chains; S3, pouring out the solution in the centrifuge tube after the desalting operation is finished, adding a first eluent into the reaction cavity, centrifuging the centrifuge tube to elute the reducing sugar chains, and obtaining a first eluent containing the reducing sugar chains in the centrifuge tube; S4, adding a ubiquitination reagent into the centrifuge tube to carry out ubiquitination reaction on the reducing sugar chain; S5, after the ubiquitination reaction is finished, adding water to terminate the reaction, transferring the liquid in the centrifuge tube into a reaction cavity, and centrifuging the centrifuge tube; S6, after the desalting operation is finished, pouring out the solution in the centrifuge tube, adding a second eluent into the reaction cavity, centrifuging the centrifuge tube to elute the reduction-ubiquitination sugar chains, and obtaining a second eluent containing the reduction-ubiquitination sugar chains in the centrifuge tube.
  6. 6. The method for reducing and ubiquitinating a sugar chain according to claim 5, wherein the first eluent is dimethyl sulfoxide.
  7. 7. The method for reducing and ubiquitination of a sugar chain according to claim 5, wherein the first eluent is dimethyl sulfoxide heated to 50-60 ℃.
  8. 8. The method for reducing and ubiquitination of a sugar chain according to claim 5, wherein the second eluent is ethyl acetate, ethyl formate, methyl acetate or methyl propionate.
  9. 9. The method for reducing and ubiquitination of sugar chains according to claim 5, wherein the sugar chain sample is a sugar chain standard, a free sugar chain, and a released sugar chain of a protein sample or a biological sample.
  10. 10. The method for reducing and ubiquitinating a sugar chain according to claim 5, wherein the reducing agent is NaBH 4 solution prepared by using an aqueous solution of NH 4 OH as a solvent, and the ubiquitinating agent is NaOH-DMSO sol solution and methyl iodide.

Description

Sugar chain reduction and ubiquitination device and method Technical Field The invention belongs to the technical field of glycobiology, and relates to a sugar chain reduction and ubiquitination device and method. Background Glycosylation modification is one of the posttranslational modifications of proteins and is widely found in higher eukaryotes. The complex sugar chains form a 'glycocalyx' structure on the cell surface and are involved in various physiological and pathological processes such as intercellular recognition, adhesion, signal transduction, molecular transport and the like. Therefore, the analysis of the protein glycosylation structure and the change thereof has important significance for biological medicine quality control, disease occurrence mechanism research and clinical diagnosis marker discovery. The complexity and heterogeneity of sugar chains lead to the analysis of sugar chains to face great challenges in terms of detection sensitivity, reproducibility, accuracy and the like, so that a sugar chain analysis method for a trace sample is urgently needed. In the prior art, the means for improving the sugar chain analysis sensitivity mainly comprises the following steps of improving the detection capability by optimizing the performance or hardware structure of a mass spectrometer, reducing the sample loss by improving the sample pretreatment flow, and enhancing the detection response of the sugar chain by chemical derivatization reaction. Although the detection sensitivity can be improved to a certain extent by the strategy based on instrument hardware transformation, the method generally requires high equipment investment, is complex to operate and maintain, has higher technical requirements on experimental conditions and operators, and is difficult to widely popularize in a conventional laboratory. In contrast, chemical derivatization is a more popular and viable strategy to increase the sensitivity of sugar chain detection. Among them, ubiquitination is a widely used sugar chain derivatization strategy. The method can remarkably improve the hydrophobicity and chemical stability of the sugar chain and remarkably improve the ionization efficiency and signal intensity of mass spectrum by methylating the hydroxyl, amino and carboxyl on the sugar chain. Meanwhile, the pan-methylation reaction can also prevent sialic acid loss, fucose molecule rearrangement and other reactions which are unfavorable for sugar group analysis. Therefore, derivatization strategies based on ubiquitination have become an important means to achieve high-sensitivity sugar chain detection and comprehensive glycogroup analysis of complex samples. The currently common pan-methylation reaction method is mainly based on Hakomori, ciucanu and the reaction mode developed by Anumula et al, and usually needs to be desalted and purified by a liquid-liquid extraction mode. However, the liquid-liquid extraction step is complicated in operation, is easy to cause sample loss and side reaction, and is unfavorable for the efficient treatment of trace samples. In order to reduce the problems, a C18 solid phase extraction column is adopted to remove and purify the ubiquitinated sugar chain, but under the high alkaline condition, the silica gel matrix is easy to dissolve, so that the desalting efficiency is reduced, and the stability of an analysis result is affected. The porous graphitized carbon (Porous Graphitic Carbon, PGC) solid phase material is considered to be suitable for desalting and enriching ubiquitously methylated sugar chains due to its good tolerance to high salt environments and strong acid and alkali conditions. However, in the prior art, the retention force of the ubimethylated sugar chains on the porous graphitized carbon material is too strong, the elution is difficult, the sample recovery rate is easy to be low, and the problem of sample loss is particularly serious especially when the sample is processed in the sub-microgram or even nanogram level. Therefore, there is still a lack of an efficient, low-loss pretreatment method suitable for ultra-trace sugar chain samples. In addition, the aldehyde group at the reducing end of the ubimethylated sugar chain can form an alpha/beta isomer, so that the alpha/beta isomer can be expressed as peak broadening or peak splitting in the chromatographic analysis process, and the detection sensitivity and the quantitative accuracy are affected. To ameliorate the above problems, the prior art generally introduces a sugar chain reduction reaction prior to ubiquitination to convert the reduced terminal aldehyde groups to sugar alcohol structures to achieve a more single and stable configuration. However, the conventional sugar chain reduction and pan-methylation are usually carried out in two independent steps, the process is complex, the reagent consumption is large, the sample loss in the operation process is remarkable, and the requirement of micro-or ultra-micro biological s