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CN-122017091-A - IgG glycopeptide selective enrichment and rapid detection method suitable for trace sample and application thereof

CN122017091ACN 122017091 ACN122017091 ACN 122017091ACN-122017091-A

Abstract

The invention discloses a selective enrichment and rapid detection method of IgG glycopeptides suitable for a trace sample and application thereof, comprising the following steps of 1) carrying out selective purification and enzymolysis on IgG in the trace sample to obtain a mixture of polypeptide and glycopeptides, 2) preparing a C18 solid-phase extraction column, and selectively eluting the glycopeptides through low-proportion organic phase conditions, or preparing a fixed absorbent cotton solid-phase extraction column, washing with 80% acetonitrile containing 1% hydrofluoric acid to remove salt and hydrophobic polypeptide, and 3) carrying out rapid liquid chromatography-mass spectrometry combined analysis on the glycopeptides after enrichment, and separating by adopting a chromatographic gradient of less than 5 minutes. The invention successfully reduces the sample demand of the IgG glycosylation analysis to a low microliter level, simplifies the experimental process, reduces the cost, improves the sensitivity, the selectivity and the repeatability of the detection, and has important scientific research value and clinical transformation application significance.

Inventors

  • LI QINGRUN
  • ZENG RONG
  • QIN PANPAN

Assignees

  • 中国科学院上海高等研究院

Dates

Publication Date
20260512
Application Date
20260120

Claims (10)

  1. 1. The IgG glycopeptide selective enrichment and rapid detection method suitable for the trace sample is characterized by comprising the following steps of: 1) Performing selective purification and enzymolysis on IgG in the trace sample to obtain a mixture of polypeptide and glycopeptide; 2) The mixture was subjected to glycopeptide enrichment and desalting using any of the following protocols: The scheme A is that a C18 solid phase extraction column consisting of a C18 membrane filler and a pipette tip is used, the mixture is loaded after methanol and 70% acetonitrile containing 0.1% hydrofluoric acid are sequentially activated, and glycopeptides are selectively eluted through low-proportion organic phase conditions, so that the desalting and enriching integration is realized; The method comprises the steps of using a shaping absorbent cotton solid-phase extraction column consisting of shaping absorbent cotton and a pipette suction head or a 96-hole column, sequentially activating the shaping absorbent cotton solid-phase extraction column by ultrapure water and 80% acetonitrile containing 1% hydrofluoric acid, loading the mixture, cleaning the mixture by using 80% acetonitrile containing 1% hydrofluoric acid to remove salt and hydrophobic polypeptide, and finally eluting glycopeptide by using 0.1% hydrofluoric acid aqueous solution to realize selective enrichment of the glycopeptide; 3) And (3) carrying out rapid liquid chromatography-mass spectrometry analysis on the glycopeptides enriched in the step (2), and separating by adopting a chromatographic gradient of less than 5 minutes.
  2. 2. The method of claim 1, wherein in scheme a, the low proportion of organic phase is 20% -30% acetonitrile comprising 0.1% hydrofluoric acid.
  3. 3. The method according to claim 1, wherein in the scheme a, the C18 membrane packing is a single-layer circular C18 membrane packing sheet having a diameter of 0.8 to 1.2mm and is packed inside a tapered end of a 200 μl low adsorption pipette tip.
  4. 4. The method according to claim 1, wherein in the scheme B, the shaped absorbent cotton is a sheet-like or column-like material having a uniform structure, uniform fiber arrangement, uniform pore diameter, 0.8 to 2.5mm in diameter, 1 to 5mm in thickness, and 0.2 to 2mg in weight.
  5. 5. The method of claim 1, wherein the rapid liquid chromatography-mass spectrometry analysis uses two secondary mass spectrometry acquisition modes: In the DDA mode, the acquisition range of the full-scanning mass spectrum is 700-2000 m/z, the MS/MS spectrogram is acquired by adopting a TopSpeed method, and the cycle time is 2 seconds; in the DIA mode, the acquisition range of the full-scanning mass spectrum is 750-1450m/z, an isolation window of 3 Th is used for MS/MS spectrogram acquisition, and the maximum injection time is 5 milliseconds; Both DDA and DIA modes use high energy collision dissociation fragmentation mode with normalized collision energy set at 30%.
  6. 6. The method of claim 1, wherein the micro clinical sample is plasma, tumor interstitial fluid or cerebrospinal fluid in a volume of 1-5 μl.
  7. 7. The method according to claim 1, wherein the selective purification and enzymolysis of IgG in step 1) comprises mixing the sample with protein G affinity material=1:2 (V: V), incubating for 60 minutes at room temperature at 800 rpm, washing 3 times with 200. Mu.L of phosphate buffer and 200. Mu.L of ultra pure water, eluting with 50. Mu.L of 0.1% hydrofluoric acid, adding 5. Mu. LpH =8.5 of 500mM tris-hydrochloric acid to the purified IgG, adjusting the pH to 7.5-8.0, denaturing at 95 ℃ for 10 minutes, returning to room temperature, adding 5mM tris (2-carboxyethyl) phosphine hydrochloride at a final concentration of 5mM, reacting for 20 minutes at room temperature, adding indole-3-acetic acid at a final concentration of 10mM at room temperature, and performing light-shielding reaction for 20 minutes, adding trypsin at an enzyme/protein=1:50 mass ratio, and performing enzymolysis at 37 ℃ overnight.
  8. 8. The method according to claim 1, wherein in the protocol A, after loading the mixture, the mixture is centrifuged at 500 Xg for 1 minute, before eluting, 100. Mu.L of 0.1% hydrofluoric acid is used to wash the salt in the sample, and the centrifugation is repeated 3 times for 1 minute, and the condition of the selective elution is that 100. Mu.L of 20% -30% acetonitrile solution containing 0.1% hydrofluoric acid is used, and 300 Xg is used to centrifuge for 1 minute, and in the protocol B, after loading the mixture, 100. Mu.L of 80% acetonitrile containing 1% hydrofluoric acid is used to remove the salt and the hydrophobic polypeptide, 100 Xg is centrifuged for 30 seconds, and 3 times is repeated, and finally 100. Mu.L of 0.1% hydrofluoric acid is used to elute glycopeptide, and 50 Xg is centrifuged for 30 seconds, and 2 times is repeated.
  9. 9. The method according to claim 1, wherein the chromatographic separation in step 3) is performed on a C18 analytical column, and the gradient of the flash liquid chromatography is such that mobile phase A is an ultrapure water solution containing 0.1% formic acid, mobile phase B is a 99.9% acetonitrile solution containing 0.1% formic acid, phase B is linearly increased to 10% at a flow rate of 3. Mu.L/min in 0 to 0.5 minutes at the beginning of 0 minutes, 10% of phase B is maintained at a flow rate of 1. Mu.L/min in 0.5 to 1.5 minutes, phase B is increased to 16% at a flow rate of 1. Mu.L/min in 1.5 to 3.5 minutes, phase B is increased to 30% at a flow rate of 3. Mu.L/min in 3.5 to 4.0 minutes, the chromatographic column is rinsed with 100% phase B at a flow rate of 3. Mu.L/min in 4.0 to 5.0 minutes, the electrospray voltage of mass spectrometry is maintained at 320℃at a capillary temperature of 20 ℃.
  10. 10. Use of the method according to any one of claims 1-9 for disease biomarker discovery and high throughput screening of clinical large cohort samples.

Description

IgG glycopeptide selective enrichment and rapid detection method suitable for trace sample and application thereof Technical Field The invention belongs to the technical field of biochemical analysis, and particularly relates to a selective enrichment and rapid detection method of IgG glycopeptides applicable to a trace sample and application thereof. Background Protein N-glycosylation is one of the most common post-translational modifications in organisms, as demonstrated by the prior art. N-glycosylation of the Fc-segment of immunoglobulin G (IgG) plays a critical role in regulating effector functions of IgG such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and anti-inflammatory activity, and is closely related to the occurrence and development of various diseases. The microenvironment is an important window for observing diseases, has extremely high scientific research and clinical value, so that the accurate and efficient analysis of IgG glycosylation in the tumor microenvironment is realized, and the method has important significance for the discovery of novel biomarkers and the exploration of disease mechanisms. The conventional IgG glycopeptide sample preparation process generally comprises a plurality of steps of IgG purification, enzymolysis, desalting, glycopeptide enrichment and the like, and is long in process and easy to cause the loss of target glycopeptides, wherein the desalting and enriching steps are the main links for causing the loss of the samples, so that the conventional method is difficult to be suitable for treating trace samples (such as interstitial fluid of tissues, the volume of which is only 1-10 mu l, and the IgG content is about 1/5-1/10 of that of plasma, cerebrospinal fluid and the like). In recent years, various integrated methods of pretreatment of a glycoprotein histology sample have been disclosed, such as the method disclosed in CN202210063100 patent for integrating a mixed ion exchange resin, a C18 membrane and a hydrophilic interaction chromatography (hiclc) packing in a single pipette tip, which enables the full-flow integration from proteolysis to specialized glycopeptide enrichment. However, the existing integration technology still cannot avoid the special enrichment step, and lacks a scheme capable of efficiently preparing IgG glycopeptides directly from a trace sample. Although such integrated materials improve the efficiency of operation to some extent, it is still unavoidable to rely on specialized glycopeptide enrichment materials such as HILIC, lectin or strong anion exchange materials, while adapting very low volume samples is difficult, and in addition, microstructural integrated materials are often costly and limited in terms of their practical controllability and versatility. In conventional enrichment protocols, the C18 solid phase extraction material is used primarily for desalination. Since glycopeptides are strongly hydrophilic and remain poorly on C18 materials, C18-mediated "desalination" and truly "glycopeptide enrichment" have been regarded as two separate steps. However, studies have shown that selective elution of glycopeptides can be achieved at very low proportions of organic phase (e.g., 1-10% ACN), suggesting that C18 materials have the potential to achieve "quasi-enrichment" of glycopeptides under specific conditions. However, the current related research focuses on peptide segment desalination or fractionation, and the optimization of the system for selective elution of glycopeptides from a low organic phase is not enough, so that the application of the system to a trace sample is rare. On the other hand, absorbent cotton has been used for simple metabolite or polypeptide adsorption experiments as a very low cost and widely available material. The hydrophilic fiber structure can absorb water-soluble or weak polar molecules, and is also theoretically suitable for glycopeptide enrichment. However, most of the materials used in the existing researches are amorphous cotton masses or cotton slivers, and the materials have the problems of nonuniform structure, inconsistent pore diameter, uncontrollable adsorption surface area and the like, so that glycopeptide enrichment repeatability is poor, impurities are easy to entrain, and the quantitative stability requirement of the glycopeptides is difficult to meet. At present, no patent or literature system utilizes absorbent cotton materials with uniform shapes and structures to enrich glycopeptides, and further lacks optimization and verification of application performance of the absorbent cotton materials in micro samples. At the analysis and detection end, a conventional liquid chromatography-mass spectrometry (LC-MS/MS) method is usually adopted to ensure the separation effect, and a long chromatographic gradient of 60-120 min is adopted, which severely limits the treatment flux of a large clinical sample array. In recent years, data Independent A