IgG glycopeptide selective enrichment and rapid detection method suitable for trace sample and application of IgG glycopeptide selective enrichment and rapid detection method

Abstract

The invention discloses an IgG glycopeptide selective enrichment and rapid detection method suitable for a trace sample and application of the IgG glycopeptide selective enrichment and rapid detection method. The IgG glycopeptide selective enrichment and rapid detection method comprises the following steps: 1) performing selective purification and enzymolysis on IgG in the trace sample to obtain a polypeptide and glycopeptide mixture; 2) a scheme A: preparing a C18 solid-phase extraction small column, and selectively eluting glycopeptide under a low-proportion organic phase condition; or the scheme B comprises the following steps: preparing a sizing degreasing cotton solid-phase extraction small column, and cleaning by using 80% acetonitrile containing 1% hydrofluoric acid to remove salt and hydrophobic polypeptide so as to realize glycopeptide enrichment; and (3) carrying out rapid liquid chromatography-mass spectrometry analysis on the enriched glycopeptides, and separating by adopting a chromatographic gradient of less than 5 minutes. According to the method, the sample demand quantity of IgG glycosylation analysis is successfully reduced to a low microliter level, the experimental process is simplified, the cost is reduced, the detection sensitivity, selectivity and repeatability are improved, and the method has important scientific research value and clinical transformation application significance.

Description

Technical Field

[0001] This invention belongs to the field of biochemical analysis technology, and more specifically relates to a method for selective enrichment and rapid detection of IgG glycopeptides suitable for trace samples and its application. Background Technology

[0002] Protein N-glycosylation is one of the most prevalent post-translational modifications in living organisms, as confirmed by existing technologies. N-glycosylation of the Fc fragment of immunoglobulin G (IgG) plays a crucial role in regulating the effector functions of IgG (such as antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and anti-inflammatory activity), and is closely related to the occurrence and development of various diseases. The tumor microenvironment, as an important window for observing diseases, possesses extremely high scientific and clinical value. Therefore, precise and efficient analysis of IgG glycosylation in the tumor microenvironment is of great significance for the discovery of novel biomarkers and the exploration of disease mechanisms.

[0003] Traditional IgG glycopeptide sample preparation typically involves multiple steps, including IgG purification, enzymatic digestion, desalting, and glycopeptide enrichment. This process is lengthy and prone to loss of target glycopeptides, with desalting and enrichment being the main causes of sample loss. This makes traditional methods unsuitable for processing micro-volume samples (such as interstitial fluid, with a volume of only 1-10 µl and an IgG content approximately 1 / 5-1 / 10 of plasma; and cerebrospinal fluid). In recent years, several integrated glycoproteomics sample pretreatment methods have been disclosed. For example, patent CN202210063100 discloses a method integrating a mixed ion exchange resin, a C18 membrane, and hydrophilic interaction chromatography (HILIC) packing material within a single pipette tip, achieving a complete workflow from enzymatic digestion to specialized glycopeptide enrichment. However, existing integrated technologies still cannot circumvent the specialized enrichment step and lack a scheme for efficiently preparing IgG glycopeptides directly from micro-volume samples. Although such integrated materials improve operational efficiency to some extent, they still inevitably rely on specialized glycopeptide enrichment materials such as HILIC, lectins, or strong anion exchange materials, and are difficult to adapt to extremely low-volume samples. In addition, microstructured integrated materials are usually expensive, and their experimental controllability and universality are also limited.

[0004] In traditional enrichment schemes, C18 solid-phase extraction materials are mainly used for desalting. Due to the strong hydrophilicity of glycopeptides, their retention on C18 materials is weak; therefore, C18-mediated "desalting" and true "glycopeptide enrichment" have traditionally been considered two separate steps. However, existing research has shown that glycopeptides can be selectively eluted under extremely low organic phase conditions (e.g., 1-10% ACN), suggesting that C18 materials have the potential to achieve "quasi-enrichment" of glycopeptides under specific conditions. However, current research largely focuses on peptide desalting or fractionation, and systematic optimization for "selective elution of glycopeptides in low organic phases" is insufficient, with its application in trace samples being even rarer.

[0005] On the other hand, absorbent cotton, as a low-cost and widely available material, has been used in simple metabolite or peptide adsorption experiments. Its hydrophilic fiber structure can adsorb water-soluble or weakly polar molecules, and theoretically, it is also suitable for glycopeptide enrichment. However, existing studies mostly use amorphous cotton balls or strips, which suffer from structural inhomogeneity, inconsistent pore sizes, and uncontrollable adsorption surface areas. This leads to poor reproducibility of glycopeptide enrichment, easy inclusion of impurities, and difficulty in meeting the quantitative stability requirements of glycoproteomics. Currently, no patents or literature systematically utilize shaped, structurally uniform absorbent cotton for glycopeptide enrichment, and there is a lack of optimization and validation of its performance in trace samples.

[0006] In analytical processing, traditional liquid chromatography-mass spectrometry (LC-MS / MS) methods often employ long chromatographic gradients of 60-120 min to ensure separation efficiency, which severely limits the throughput of large clinical cohort samples. In recent years, data-independent acquisition (DIA) has gradually become the mainstream method in deep proteomics, but its development in the field of glycopeptide analysis remains relatively slow. In particular, achieving compatibility between short gradients (e.g., 5 min) and DIA mode places higher demands on the purity, enrichment effect, and background ion control of sample pretreatment.

[0007] Therefore, there is an urgent need in this field for an experimental scheme that can adapt to trace samples, simplify experimental procedures, and improve the efficiency and throughput of glycopeptide detection. Summary of the Invention

[0008] The purpose of this invention is to provide a selective enrichment and rapid detection method for IgG glycopeptides suitable for trace samples and its application, thereby solving the defects of existing technologies, such as lengthy processes that easily lead to loss of glycopeptides in trace samples, reliance on special enrichment materials and high costs, insufficient universality and controllability, low glycopeptide detection efficiency and low analytical throughput.

[0009] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0010] According to a first aspect of the present invention, a method for selective enrichment and rapid detection of IgG glycopeptides suitable for trace samples is provided, comprising the following steps:

[0011] 1) Selective purification and enzymatic digestion of IgG in trace clinical samples yielded a mixture of polypeptides and glycopeptides;

[0012] 2) The mixture is subjected to glycopeptide enrichment and desalting using any of the following methods:

[0013] Option A: A C18 solid-phase extraction column composed of C18 membrane packing and pipette tips is used. After sequential activation with methanol and 70% acetonitrile containing 0.1% hydrofluoric acid, the mixture is loaded and selectively eluted with glycopeptides under low-proportion organic phase conditions to achieve integrated desalting and enrichment.

[0014] Option B: A solid-phase extraction column consisting of shaped defatted cotton and pipette tips or a 96-well column is used. After being activated sequentially with ultrapure water and 80% acetonitrile containing 1% hydrofluoric acid, the mixture is loaded onto the column. Then, it is washed with 80% acetonitrile containing 1% hydrofluoric acid to remove salts and hydrophobic peptides. Finally, it is washed with 0.1% hydrofluoric acid aqueous solution to remove glycopeptides, thereby achieving selective enrichment of glycopeptides.

[0015] 3) The enriched glycopeptides were analyzed by rapid liquid chromatography-mass spectrometry, and the separation was performed using a chromatographic gradient of less than 5 minutes.

[0016] Preferably, in Scheme A, the low-proportion organic phase is 20%-30% acetonitrile and contains 0.1% hydrofluoric acid as an additive.

[0017] Preferably, in Scheme A, the C18 membrane packing is a single-layer circular C18 membrane packing sheet with a diameter of 0.8-1.2 mm, and is filled inside the conical end of a 200 μL low-adsorption pipette tip.

[0018] Preferably, in Scheme B, the shaped degreased cotton is a sheet or columnar material with uniform structure, uniform fiber arrangement, consistent pore size, diameter of 0.8-2.5mm, thickness of 1-5mm, and weight of 0.2-2mg.

[0019] Preferably, the rapid liquid chromatography-mass spectrometry (LC-MS) analysis employs the following two-stage mass spectrometry acquisition mode:

[0020] In data-dependent acquisition (DDA) mode, the acquisition range of full scan mass spectrometry is 700-2000 m / z, and the TopSpeed ​​method is used for MS / MS spectrum acquisition with a cycle time of 2 seconds.

[0021] In data-independent acquisition (DIA) mode, the acquisition range of full-scan mass spectrometry is 750-1450 m / z, and MS / MS spectrum acquisition uses a 3 Th isolation window with a maximum injection time of 5 milliseconds;

[0022] Both DDA and DIA modes use high-energy collisional dissociation (HCD) fragmentation, with the normalized collision energy (NCE) set to 30%.

[0023] Preferably, the trace clinical sample is 1-5 μL of plasma, tumor interstitial fluid, or cerebrospinal fluid.

[0024] Preferably, the selective purification and enzymatic digestion of IgG in step 1) specifically includes: mixing sample:protein G affinity material = 1:2 (V:V), incubating at 800 rpm for 60 minutes at room temperature, washing three times each with 200 μL phosphate buffer and 200 μL ultrapure water, and eluting with 50 μL 0.1% hydrofluoric acid to obtain purified IgG; adding 5 μL 500 mM tris(hydroxymethyl)aminomethane-hydrochloric acid (pH=8.5) to the purified IgG to adjust the pH to 7.5-8.0, denaturing at 95°C for 10 minutes, and then restoring to room temperature, adding 5 mM tris(2-carboxyethyl)phosphine hydrochloride and reacting at room temperature for 20 minutes, then adding 10 mM indole-3-acetic acid and reacting at room temperature in the dark for 20 minutes, and finally adding trypsin at a mass ratio of enzyme:protein = 1:50 and digesting overnight at 37°C.

[0025] Preferably, in Scheme A, after loading the mixture, centrifuge at 500×g for 1 minute; before elution, wash the sample with 100 μL of 0.1% hydrofluoric acid to remove salts, centrifuge at 500×g for 1 minute, and repeat 3 times; the selective elution conditions are: use 100 μL of 20%-30% acetonitrile solution containing 0.1% hydrofluoric acid, centrifuge at 300×g for 1 minute; in Scheme B, after loading the mixture, use 100 μL of 80% acetonitrile containing 1% hydrofluoric acid to remove salts and hydrophobic peptides, centrifuge at 100×g for 30 seconds, repeat 3 times, and finally use 100 μL of 0.1% hydrofluoric acid to elute glycopeptides, centrifuge at 50×g for 30 seconds, and repeat 2 times.

[0026] Preferably, the specific gradient for rapid liquid chromatography in step 3) is as follows: mobile phase A is an ultrapure aqueous solution containing 0.1% formic acid, and mobile phase B is a 99.9% acetonitrile solution containing 0.1% formic acid; initially, phase B is 5% at 0 minutes, linearly increasing to 10% at a flow rate of 3 μL / min within 0–0.5 minutes, maintaining 10% phase B at a flow rate of 1 μL / min within 0.5–1.5 minutes, increasing phase B to 16% at a flow rate of 1 μL / min within 1.5–3.5 minutes, increasing phase B to 30% at a flow rate of 3 μL / min within 3.5–4.0 minutes, and rinsing the column with 100% phase B at a flow rate of 3 μL / min within 4.0–5.0 minutes; the electrospray voltage for mass spectrometry analysis is 1.9 kV, and the capillary temperature is maintained at 320°C.

[0027] According to a second aspect of the present invention, an application of the above-described method in the discovery of disease biomarkers or in high-throughput screening of large clinical cohort samples is provided.

[0028] Both enrichment schemes provided by this invention are fully compatible with the pre- and post-processing workflows of this invention, and are adapted to different experimental scenarios based on their core advantages:

[0029] C18 Low Organic Phase Enrichment Method: Its core advantage lies in its highly integrated process, which requires no additional special enrichment materials, is simple and efficient to operate, and is suitable for rapid detection scenarios that require analysis speed and direct quantification.

[0030] The fixed-size absorbent cotton enrichment method is characterized by low cost and high reproducibility, making it an excellent alternative for enriching highly hydrophilic glycopeptides, especially suitable for research scenarios with higher selectivity requirements for glycopeptide enrichment.

[0031] According to the research findings of this invention, in Scheme A, a 20%-30% acetonitrile solution containing 0.1% hydrofluoric acid is the optimal glycopeptide elution condition. This condition not only ensures a high number of glycopeptides identified but also has excellent elution selectivity, which can meet the technical requirements of integrated desalting and enrichment of C18 solid phase extraction columns, and provides key parameter support for the efficient enrichment of IgG glycopeptides in trace samples.

[0032] The study also found that in Scheme B, with the thickness of the absorbent cotton fixed at 1 mm, the diameter of the absorbent cotton was positively correlated with the quantitative performance of glycopeptides; the larger the diameter, the better the adsorption and recovery effect of glycopeptides. Considering the convenience of experimental operation and cost control, it is recommended to choose absorbent cotton with a diameter of 1.6-2.0 mm (corresponding to a weight of 1.0-1.5 mg). This can ensure high quantitative performance while avoiding increased flow resistance or material waste caused by excessive diameter, and is suitable for the efficient enrichment and accurate quantification of IgG glycopeptides in trace samples.

[0033] Validation using 23 pairs of clinical colorectal cancer samples confirmed that the method of this invention can accurately capture differences in IgG N-glycosylation from samples of different sources (plasma, tumor stromal fluid, and adjacent normal stromal fluid), and possesses high sensitivity and high stability. The experimental results provide direct data support for the screening of colorectal cancer-related IgG N-glycosylation biomarkers, and also validate the practicality and effectiveness of the method in disease mechanism research and large-scale clinical cohort sample screening, meeting the needs of clinical translational applications.

[0034] The present invention provides a method for selective enrichment and rapid detection of IgG glycopeptides in trace samples, and its application therein, which has the following advantages over the prior art:

[0035] 1. Ultra-high sensitivity and broad sample compatibility. Through end-to-end optimization, including IgG purification, glycopeptide enrichment, and mass spectrometry detection parameter adaptation, the initial sample volume required for analysis has been successfully reduced to a low micro-liter level (1-5 μL, typically 5 μL). This breakthrough enables for the first time precise analysis of IgG glycosylation in precious trace samples such as plasma, tumor interstitial fluid (IgG content is only 1 / 5-1 / 10 of plasma), and cerebrospinal fluid, overcoming the technical bottleneck of traditional methods that cannot be adapted to such samples due to large sample requirements and high loss rates.

[0036] 2. Process Integration: C18 Low-Organic-Phase Elution Achieves Integrated Desalination and Enrichment. This innovative approach utilizes the stronger hydrophilicity of glycopeptides compared to non-glycopeptides to establish a low-organic-phase selective elution scheme on conventional C18 solid-phase materials, integrating the traditionally separate two-step processes of desalination and glycopeptide enrichment into a single step. Through systematic optimization of the acetonitrile ratio (comparing gradients of 20%, 30%, 50%, and 90%), the optimal elution system of 20%-30% acetonitrile + 0.1% hydrofluoric acid was determined. This system maximizes the elution of glycopeptides while minimizing the entrainment of non-glycopeptides, simultaneously removing salts. This scheme eliminates the need for additional specialized enrichment materials, significantly simplifying the experimental procedure, reducing sample loss, and improving operational efficiency and result stability.

[0037] 3. Low Cost and High Stability: A Highly Efficient Glycopeptide Enrichment Solution Using Shaped Absorbent Cotton. This study is the first to utilize shaped absorbent cotton with a uniform structure and controllable parameters (diameter 0.8-2.5 mm, thickness 1-5 mm, weight 0.2-2 mg) as a glycopeptide enrichment material, overcoming the shortcomings of traditional amorphous cotton clumps / strips, such as uneven structure, uncontrollable adsorption area, and poor reproducibility. Leveraging its uniform fiber arrangement and stable pore distribution, it significantly improves the reproducibility and quantitative stability of glycopeptide enrichment. Simultaneously, the material is extremely low-cost and widely available, fully adaptable to large-scale clinical sample applications, providing a cost-effective alternative for glycopeptide enrichment.

[0038] 4. High-throughput analysis: Adapted to the needs of large-scale clinical screening. The highly efficient glycopeptide enrichment step significantly reduces sample complexity, successfully achieving compatibility with 5-minute ultrafast liquid chromatography gradient and data-independent acquisition (DIA) mass spectrometry modes. Compared to traditional 60-120 minute long gradient analyses, this invention shortens the single-sample analysis cycle by more than 90%, and the DIA mode can identify 304 glycopeptide precursor ions and quantify 164 glycopeptide precursor ions, far exceeding the detection efficiency of traditional DIA modes. The entire workflow achieves rapid transformation from sample processing to result output, meeting the needs of high-throughput screening and disease biomarker screening for large-scale clinical samples.

[0039] In summary, this invention, through the innovative integration of a C18 low-organic-phase elution desalting and enrichment process with a low-cost, high-stability enrichment strategy using shaped absorbent cotton, successfully reduces the sample volume required for IgG glycosylation analysis to a low-micro-level. It overcomes the technical bottleneck of traditional methods being unable to adapt to precious micro-samples such as plasma and tumor stromal fluid. Furthermore, by leveraging the compatibility of a 5-minute ultrafast liquid chromatography gradient with DIA mass spectrometry mode, it achieves high-throughput analysis, simplifying the experimental procedure, reducing costs and sample loss, and improving the sensitivity, selectivity, and repeatability of detection. This provides an efficient and flexible technical solution for glycoproteomics research and strongly supports the screening of disease biomarkers and the exploration of disease mechanisms in large clinical cohorts, demonstrating significant scientific research value and clinical translational application significance. Attached Figure Description

[0040] Figure 1 is a flowchart of the glycopeptide enrichment process of the present invention: where A is the C18 low organic phase enrichment method (i.e., scheme A); B is the shaped defatted cotton enrichment method (i.e., scheme B); 1 is a pipette tip; 2 is a C18 membrane packing material; solution 1 is a mixture of 0.1% hydrofluoric acid and 99.9% ultrapure water; solution 2 is a mixture of acetonitrile, 0.1% hydrofluoric acid and ultrapure water in appropriate low proportions; 3 is a shaped defatted cotton sheet; 4 is a shaped defatted cotton solid-phase extraction column formed by filling 200 μL low adsorption pipette tips with shaped defatted cotton; 5 is a shaped defatted cotton solid-phase extraction column formed by filling the wells of a 96-well SPEPlate plate with shaped defatted cotton.

[0041] Figure 2 is a comparison of the effects of different first-order mass spectrometry acquisition ranges on the identification of IgG glycopeptides. The vertical axis represents the number of glycopeptide precursor ions, and the horizontal axis represents the three different mass spectrometry acquisition ranges, namely 380-980 m / z, 380-1450 m / z, and 750-1450 m / z.

[0042] Figure 3 is a comparison of the qualitative and quantitative performance of the data-dependent acquisition mode (DDA) and the data-independent acquisition mode (DIA). The horizontal axis corresponds to the DDA mode and the DIA mode, respectively, and the vertical axis shows the number of glycopeptide precursor ions from the two dimensions of qualitative identification and quantitative analysis.

[0043] Figure 4 shows the identification results of IgG glycopeptides under different elution conditions. The eluents all contained 0.1% hydrofluoric acid, with only the acetonitrile ratio differing (20%, 30%, 50%, and 90%, respectively). In the figure, A is a comparison of the number of glycopeptide precursor ions identified under different elution conditions; B is an intersection analysis of the identification results under different elution conditions, i.e., the overlap and specificity of glycopeptide precursor ions identified by eluents with different acetonitrile ratios.

[0044] Figure 5 shows the quantitative performance evaluation of glycopeptides on 1 mm thick, defatted cotton with different diameters; where A is a schematic diagram of solid phase extraction columns composed of defatted cotton with different diameters; B is a comparison of the overall quantitative performance of defatted cotton with different diameters; and C is the difference in quantitative results of three IgG glycopeptides with different abundances on defatted cotton with different diameters.

[0045] Figure 6 shows the results of partial least squares discriminant analysis (PLS-DA), illustrating the differences in the expression of IgG glycopeptides in the plasma, tumor stroma fluid, and adjacent stroma fluid of 23 pairs of colorectal cancer patients. Detailed Implementation

[0046] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the techniques used in the embodiments are conventional practices in the art, or experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, the reagents and materials used in the embodiments are commercially available.

[0047] Example 1: Preparation of IgG peptide samples (common pretreatment basis for subsequent glycopeptide enrichment)

[0048] This embodiment provides a standardized preparation process for IgG peptide samples, providing a unified pretreatment basis for subsequent glycopeptide enrichment methods. The specific steps are as follows:

[0049] 1. Selective purification of IgG: IgG in trace clinical samples was specifically purified using protein G affinity material (protein G agarose gel, rapid flow, catalog number P3296, purchased from Merck). The sample was mixed with protein G affinity material at a ratio of 1:2 (V:V) and incubated at 800 rpm for 60 minutes at room temperature to allow IgG to fully bind to protein G. The sample was then washed three times each with 200 μL phosphate buffer and 200 μL ultrapure water to remove non-specifically bound impurities. Finally, the IgG bound to protein G was eluted with 50 μL 0.1% hydrofluoric acid to obtain a purified IgG solution.

[0050] 2. Protein denaturation, reduction, and alkylation: Add 5 μL of 500 mM tris(hydroxymethyl)aminomethane-hydrochloric acid buffer (pH=8.5) to the purified IgG solution to adjust the pH of the system to 7.5-8.0; then denature at 95°C for 10 minutes and allow to cool naturally to room temperature; add 5 mM tris(2-carboxyethyl)phosphine hydrochloride to the system and react at room temperature for 20 minutes to reduce disulfide bonds; then add 10 mM indole-3-acetic acid and react at room temperature in the dark for 20 minutes to complete the alkylation process and avoid disulfide bond renaturation.

[0051] 3. Trypsin hydrolysis: Add trypsin to the above-treated system at a mass ratio of 1:50 (enzyme to protein) and incubate overnight at 37°C to obtain a mixed solution of polypeptides and glycopeptides, which will be used for subsequent glycopeptide enrichment and desalting steps.

[0052] Example 2: Two methods were used for glycopeptide enrichment and desalting.

[0053] This embodiment uses the polypeptide and glycopeptide mixed sample prepared in Example 1. Glycopeptide enrichment and desalting were performed using the C18 low organic phase enrichment method (Scheme A) and the fixed defatted cotton enrichment method (Scheme B), respectively, to verify the feasibility and enrichment effect of the two schemes. The specific steps are as follows:

[0054] 1. Preliminary preparations

[0055] Take 1-5 μL of a small amount of clinical sample (such as plasma or tumor tissue interstitial fluid) and process it according to the standardized procedure in Example 1 to obtain a mixed sample of peptides and glycopeptides, which will be used as raw material for subsequent enrichment experiments.

[0056] 2. Scheme A: C18 Low Organic Phase Enrichment Method

[0057] (1) Preparation of C18 solid phase extraction column

[0058] Empore™ C18 membrane material (product number: 2215) was selected and cut into circular packing sheets with a diameter of 1.0 mm (single-layer packing). The packing sheets were then placed inside the conical end of a 200 μL low-adsorption pipette tip to prepare a self-made C18 solid-phase extraction column.

[0059] (2) Column activation

[0060] The C18 solid-phase extraction column was activated successively with methanol and 70% acetonitrile (containing 0.1% hydrofluoric acid) to ensure that the packing material was fully wetted.

[0061] (3) Sample loading and desalination

[0062] The previously prepared polypeptide and glycopeptide mixture sample was loaded onto an activated C18 solid-phase extraction column and centrifuged at 500×g for 1 minute to ensure full contact between the sample and the C18 packing material. The column was then washed with 100 μL of 0.1% hydrofluoric acid to remove salts, and centrifuged at 500×g for 1 minute. This washing step was repeated 3 times to thoroughly remove impurities and salts from the system.

[0063] (4) Selective elution of glycopeptides

[0064] Using an optimized 100 μL eluent (a 20%-30% acetonitrile solution containing 0.1% hydrofluoric acid), the glycopeptides adsorbed on the C18 packing were selectively eluted by centrifugation at 300×g for 1 minute. The eluent was collected, completing the integrated enrichment and desalting of glycopeptides. The optimal ratio of acetonitrile in this eluent was verified by comparative experiments in Example 4.

[0065] 3. Option B: Enrichment method using shaped absorbent cotton

[0066] (1) Preparation and filling of shaped degreased cotton

[0067] Ordinary absorbent cotton is compressed into a uniform sheet shape (basic size: 300 mm × 300 mm, thickness 1-5 mm optional), and then laser-cut to obtain circular cotton sheets of a fixed diameter (diameter 0.8-2.5 mm optional, single sheet weight 0.2-2 mg); according to the experimental throughput requirements, the circular cotton sheets are loaded into 200 μL low-absorption pipette tips (e.g., Figure 1 In the wells of a 96-well SPE plate (as shown in Figure 4) or 96-well SPE plate (as shown in Figure 5), a shaped degreased cotton solid phase extraction column is formed.

[0068] (2) Activation of enrichment units

[0069] The shaped degreased cotton was activated by successively using 200 μL of ultrapure water and 80% acetonitrile (containing 1% hydrofluoric acid). Each step was repeated 3 times, and each time the cotton was centrifuged at 100×g for 30 seconds to ensure that the cotton was fully activated and potential impurities were removed.

[0070] (3) Sample loading and impurity removal

[0071] The previously prepared peptide and glycopeptide mixture sample was loaded onto activated, defatted cotton, and then the enrichment unit was washed with 100 μL of 80% acetonitrile (containing 1% hydrofluoric acid). The mixture was centrifuged at 100×g for 30 seconds and repeated 3 times to remove salts and impurities such as hydrophobic peptides from the system.

[0072] (4) Glycopeptide elution

[0073] Using 100 μL of 0.1% hydrofluoric acid as the eluent, the glycopeptides were eluted by centrifugation at 50×g for 30 seconds. The elution was repeated twice, and the two eluents were combined to complete the glycopeptide enrichment.

[0074] Example 3: The effect of different mass-to-charge ratio acquisition ranges of primary mass spectrometry on the identification of IgG glycopeptides

[0075] This embodiment aims to screen the optimal mass-to-charge ratio (m / z) acquisition range for the identification of trace IgG glycopeptides using primary mass spectrometry, providing parameter support for subsequent high-efficiency detection. The specific experimental procedure and results are as follows:

[0076] 1. Experimental Sample Preparation

[0077] Take a small amount of clinical sample (1-5 μL, such as plasma or tumor tissue interstitial fluid), and prepare and enrich glycopeptide samples according to the standardized procedures in Examples 1 and 2.

[0078] 2. Experimental Instruments and Parameter Settings

[0079] Analysis was performed using a Vanquish ultra-high performance liquid chromatography system (Thermo Scientific) coupled with an Orbitrap Astral mass spectrometer (Thermo Scientific). Except for the primary mass spectrometry acquisition range, the remaining liquid chromatography and mass spectrometry parameters are as follows:

[0080] Chromatographic conditions: A 7 cm long, 150 μm inner diameter C18 analytical column was used; mobile phase A was an ultrapure aqueous solution containing 0.1% formic acid, and mobile phase B was a 99.9% acetonitrile solution containing 0.1% formic acid; a short gradient elution was used for 5 minutes, specifically: 5% B phase at 0 minutes, linearly increased to 10% at a flow rate of 3 μL / min from 0 to 0.5 minutes, maintained at 10% B phase (flow rate of 1 μL / min) from 0.5 to 1.5 minutes, increased to 16% B phase at a flow rate of 1 μL / min from 1.5 to 3.5 minutes, increased to 30% at a flow rate of 3 μL / min from 3.5 to 4.0 minutes, and the column was washed with 100% B phase at a flow rate of 3 μL / min from 4.0 to 5.0 minutes.

[0081] Mass spectrometry conditions: positive ion mode detection, electrospray voltage 1.9 kV, capillary temperature 320℃; secondary mass spectrometry adopted data-independent acquisition (DIA) mode, using high-energy collisional dissociation (HCD) fragmentation, normalized collision energy (NCE) set to 30%, MS / MS spectrum acquisition adopted 3 Th isolation window, and maximum injection time 5 ms.

[0082] 3. Experimental Variable Design

[0083] Three different full-scan acquisition ranges for first-order mass spectrometry were set up: 380-980 m / z, 380-1450 m / z, and 750-1450 m / z, with all other parameters kept constant. The experiments were conducted in parallel to compare and identify the results.

[0084] 4. Experimental Results and Analysis

[0085] The experimental results are shown in Figure 2: When the primary mass spectrometry acquisition range was 380-980 m / z, only one IgG glycopeptide precursor ion was identified; when the acquisition range was expanded to 380-1450 m / z, the number of identified IgG glycopeptide precursor ions significantly increased to 136; and when the acquisition range was 750-1450 m / z, the number of identified IgG glycopeptide precursor ions reached 207, which is an increase of 206% and 52.2% compared with the previous two groups, respectively.

[0086] These results indicate that the mass-to-charge ratio (M / Z) of IgG glycopeptides is mainly concentrated in the 750-1450 m / z range. Acquisition ranges with excessively low overall M / Z (e.g., 380-980 m / z) will miss most glycopeptide signals. Conversely, when including low M / Z ranges (e.g., 380-1450 m / z), low M / Z non-glycopeptide signals may interfere with glycopeptide detection, leading to decreased identification efficiency. Therefore, 750-1450 m / z is the optimal primary mass spectrometry acquisition range for the method of this invention, enabling efficient identification of IgG glycopeptides.

[0087] Example 4: Comparison of Identification and Quantification of IgG Glycopeptides Using DDA and DIA Modes

[0088] Based on the optimal first-stage mass spectrometry acquisition range (750-1450 m / z) determined in Example 3, this embodiment further compares the identification efficiency and quantification performance of data-dependent acquisition (DDA) and data-independent acquisition (DIA) mass spectrometry modes for trace IgG glycopeptides, and screens the optimal mass spectrometry acquisition mode suitable for the method of this invention. The specific experimental procedure and results are as follows:

[0089] 1. Experimental Sample Preparation

[0090] Take 1-5 μL of a small clinical sample (such as plasma or tumor tissue interstitial fluid) and prepare and enrich the glycopeptide sample according to the standardized procedures in steps 1 and 2.

[0091] 2. Experimental Instruments and Parameter Settings

[0092] A Vanquish ultra-high performance liquid chromatography system (Thermo Scientific) was used in conjunction with an Orbitrap Astral mass spectrometer (Thermo Scientific), and the liquid chromatography parameters were kept consistent with those in Example 3.

[0093] The specific parameter settings for the two acquisition modes are as follows:

[0094] DDA mode: Full scan mass spectrometry acquisition range m / z 700–2000, using TopSpeed ​​method for MS / MS spectrum acquisition, with a cycle time of 2 seconds;

[0095] DIA mode: MS / MS spectrum acquisition uses a 3-Th isolation window, with a maximum injection time of 5 milliseconds.

[0096] 3. Experimental Variable Design

[0097] Two parallel experiments were set up, with only the secondary mass spectrometry acquisition mode changed (DDA mode and DIA mode, respectively), and all other experimental conditions kept exactly the same. The performance difference between the two modes was evaluated by comparing the number of glycopeptides identified and quantified in the two experiments.

[0098] 4. Experimental Results and Analysis

[0099] The experimental results are shown in Figure 3. Regarding identification performance, the DDA mode identified 269 IgG glycopeptide precursor ions, while the DIA mode identified 304, a 13% increase compared to the DDA mode. This indicates that the DIA mode has a wider coverage of glycopeptides and can effectively capture more low-abundance glycopeptide signals. In terms of quantitative performance, the DDA mode could only quantitatively analyze 91 glycopeptide precursor ions, while the DIA mode could quantify 164, representing an 80.2% improvement in quantitative capability.

[0100] These results demonstrate that the DIA mode, through its independent data acquisition method, avoids the problem of missed detection of low-abundance glycopeptides caused by signal competition in the DDA mode, while also exhibiting superior quantitative stability and reproducibility. Considering the application scenarios of this invention for trace samples, the DIA mode can more fully extract glycopeptide information, meeting the requirements for high-sensitivity identification and accurate quantification, making it a superior spectroscopic acquisition mode suitable for this method.

[0101] Example 5: Comparison of elution of IgG glycopeptides by different proportions of acetonitrile containing 0.1% hydrofluoric acid

[0102] This embodiment aims to screen the optimal elution conditions for glycopeptides in C18 solid-phase extraction column enrichment. By comparing the elution effects of different acetonitrile ratios containing 0.1% hydrofluoric acid on IgG glycopeptides, the optimal elution system that balances the quantity and selectivity of glycopeptide identification is identified. The specific experimental procedure and results are as follows:

[0103] 1. Experimental Sample Preparation

[0104] Take 1-5 μL of a small amount of clinical sample (such as plasma or tumor tissue interstitial fluid) and prepare and enrich glycopeptide samples according to the standardized procedures in Examples 1 and 2.

[0105] 2. Experimental Instruments and Parameter Settings

[0106] The analysis was performed using a Vanquish ultra-high performance liquid chromatography system (Thermo Scientific) coupled with an Orbitrap Astral mass spectrometer (Thermo Scientific), with all instrument parameters set to the optimal conditions determined in previous experiments.

[0107] 3. Experimental Variable Design

[0108] Four parallel elution experiments were set up, with the only variable being the volume ratio of acetonitrile in the eluent. All four eluents contained 0.1% hydrofluoric acid, and the acetonitrile ratios were 20%, 30%, 50%, and 90%, respectively. Each group used 100 μL of the corresponding eluent, and glycopeptides on the C18 column were eluted by centrifugation at 300×g for 1 minute. The eluent was collected, concentrated under vacuum, and redissolved in 5 μL of 0.1% formic acid before mass spectrometry analysis. All other experimental conditions were completely consistent.

[0109] 4. Experimental Results and Analysis

[0110] The experimental results are shown in Figure 4:

[0111] Figure 4A shows the comparison of the number of IgG glycopeptide precursor ions identified under different elution conditions. The results show that the number of glycopeptide precursor ions identified in the 20% acetonitrile group and the 30% acetonitrile group is significantly higher than that in the 50% acetonitrile group and the 90% acetonitrile group. The number of glycopeptide precursor ions identified in the 20% acetonitrile group and the 30% acetonitrile group is similar and at a relatively high level, while the number of glycopeptide precursor ions identified in the 50% acetonitrile group is significantly lower, and the number of glycopeptide precursor ions identified in the 90% acetonitrile group is the lowest. This indicates that a high proportion of acetonitrile will lead to a decrease in the elution selectivity of glycopeptides and may also carry more hydrophobic non-glycopeptide impurities, interfering with the detection of glycopeptides.

[0112] Figure 4B shows the intersection analysis of identification results under different elution conditions. The numbers in the figure represent the number of glycopeptide precursor ions specific to the corresponding elution condition (i.e., glycopeptide precursor ions that can only be identified in that group): the 20% acetonitrile group has 35 unique glycopeptide precursor ions, the 30% acetonitrile group has 34 unique glycopeptide precursor ions, while the 50% acetonitrile and 90% acetonitrile groups have only single-digit numbers of unique glycopeptides, and the intersection with the first two groups is small. This result indicates that the elution buffer with a 20%-30% acetonitrile ratio has stronger selectivity for glycopeptides and can specifically elute more target glycopeptides, while the high-ratio acetonitrile elution buffer has poor selectivity and is difficult to effectively distinguish between glycopeptides and non-glycopeptides.

[0113] Example 6: Comparison of IgG glycopeptide quantification performance of 1mm thick degreased cotton with different diameters

[0114] This embodiment aims to investigate the effects of different diameters of pre-treated absorbent cotton on the enrichment and quantification performance of IgG glycopeptides when the thickness is fixed at 1 mm, and to clarify the optimal diameter range of pre-treated absorbent cotton suitable for the analysis of glycopeptides in trace samples. The specific experimental procedure and results are as follows:

[0115] 1. Experimental Sample Preparation

[0116] Take 1 μL of human plasma sample and prepare glycopeptide precursor sample according to the standardized procedure in Example 1.

[0117] 2. Preparation of experimental materials

[0118] (1) Preparation and filling of shaped degreased cotton

[0119] Ordinary degreased cotton is selected and pressed into sheet material with uniform structure (basic size 300mm×300mm, thickness fixed at 1mm). Then, circular cotton sheets of different diameters are obtained by laser cutting, with diameter gradients set to 0.8mm, 1.0mm, 1.2mm, and 1.4mm.

[0120] Different diameter circular cotton pads were filled into the inner side of the conical end of the tip of a 200μL low-absorption pipette. Due to the compatibility between the conical structure of the pipette tip and the diameter of the cotton pad, all diameter cotton pads could be stably kept in the designated position on the pipette tip (avoiding displacement during liquid flow) and ensuring uniform flow resistance after filling, without affecting subsequent centrifugation operations.

[0121] (2) Comparison settings

[0122] Four parallel experiments were set up, with each group using a different diameter of pre-shaped absorbent cotton. All other experimental conditions were kept identical to ensure that only the diameter was the unique variable.

[0123] 3. Glycopeptide enrichment process (based on the fixed absorbent cotton enrichment method)

[0124] Each experiment was conducted according to the standard procedure of Scheme B in Example 2.

[0125] 4. Instrument and Parameter Settings

[0126] The analysis was performed using a Vanquish ultra-high performance liquid chromatography system (Thermo Scientific) coupled with an Orbitrap Astral mass spectrometer (Thermo Scientific), with the parameters being the optimal conditions determined in previous experiments.

[0127] 5. Experimental Results and Analysis

[0128] The experimental results are shown in Figure 5:

[0129] Figure 5A shows the loading status of pre-shaped absorbent cotton of different diameters in a 200μL pipette tip. It can be seen that cotton pads of various diameters are stably fixed at the conical end of the pipette tip without displacement or blockage, ensuring smooth liquid flow. Figure 5B compares the overall quantitative performance of pre-shaped absorbent cotton of different diameters. The results show that as the diameter of the pre-shaped absorbent cotton increases from 0.8mm to 1.4mm, the overall quantitative value of glycopeptides shows a continuous upward trend. This is because the increased diameter leads to a simultaneous increase in the effective adsorption area and pore capacity of the cotton pad, significantly improving the adsorption capacity for hydrophilic glycopeptides and thus increasing the glycopeptide recovery rate. Figure 5C (quantification of three different abundances of IgG glycopeptides) shows that when high, medium, and low abundance target glycopeptides are analyzed separately, their quantitative values ​​all show a consistent upward trend with the increase of the absorbent cotton diameter, and the quantitative increase of low abundance glycopeptides is more significant. This indicates that increasing the diameter of the defatted cotton not only improves the overall glycopeptide recovery rate, but also enhances the capture ability of low-abundance glycopeptides, further verifying the quantitative stability brought about by the structural uniformity of the shaped defatted cotton.

[0130] Example 7:23 Differential analysis of IgG N-glycosylation in colon cancer-related samples

[0131] This embodiment aims to verify the practical application value of the method of the present invention in the discovery of disease biomarkers. By analyzing the differences in IgG N-glycosylation profiles between plasma, tumor stromal fluid, and adjacent normal tissue stromal fluid of colorectal cancer patients, the specific changes in IgG glycosylation in the tumor microenvironment are explored. The specific experimental procedure and results are as follows:

[0132] 1. Source of experimental samples

[0133] Clinical samples from 23 patients with colorectal cancer were selected, including: plasma samples; tumor tissue interstitial fluid; and adjacent normal tissue interstitial fluid. 5 μL of each type of sample was used for the experiment. All samples complied with clinical ethical guidelines and the informed consent of the sample providers was obtained.

[0134] 2. Experimental Sample Processing and Enrichment

[0135] All samples were processed according to the standardized procedures in Examples 1 and 2.

[0136] 3. Instrument and Parameter Settings

[0137] The analysis was performed using a Vanquish ultra-high performance liquid chromatography system (Thermo Scientific) coupled with an Orbitrap Astral mass spectrometer (Thermo Scientific), with the parameters being the optimal conditions determined in previous experiments.

[0138] 4. Data Processing and Analysis Methods

[0139] The raw data were analyzed using professional mass spectrometry data analysis software to identify IgG glycopeptide precursor ions; the screening threshold was set at at least 50% of the samples in the same group having stable quantitative values, and quantification was performed down to 100 glycopeptide precursor ions; multivariate statistical analysis of the glycosylation spectra of the three groups of samples was performed by partial least squares discriminant analysis (PLS-DA) to assess the differences between groups.

[0140] 5. Experimental Results and Analysis

[0141] The experimental results are shown in Figure 6:

[0142] Figure 6A (IgG glycopeptide identification and quantification results): This figure shows the overview of IgG glycopeptide identification and quantification in three groups of samples. The results show that a total of 401 IgG glycopeptide precursor ions were identified in all samples, covering glycopeptides related to the main N-glycosylation sites of the IgG Fc segment. After screening, 100 glycopeptide precursor ions met the condition that at least 50% of the samples in the same group had quantitative values, and could be used for inter-group difference analysis.

[0143] Figure 6B (PLS-DA score plot): This plot visually displays the differences in IgG N-glycosylation profiles among the three groups of samples using partial least squares discriminant analysis. The horizontal axis represents component 1 (explanation rate 19.3%), and the vertical axis represents component 2 (explanation rate 2.6%). The results show that the adjacent normal stromal fluid group and the tumor stromal fluid group are significantly separated on the component 2 axis, suggesting that IgG N-glycosylation in the tumor microenvironment has undergone specific remodeling, which may be related to abnormal immune regulation during tumor development. The plasma group samples are clearly separated from both the adjacent normal stromal fluid group and the tumor stromal fluid group, indicating that there is an essential difference between the IgG N-glycosylation profile in the circulatory system and the local tumor microenvironment, and also verifying the unique value of the tumor microenvironment as a disease observation window. The small dispersion of samples within the same group indicates that the method of this invention has good reproducibility and the quantitative results are stable and reliable.

[0144] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Various variations can be made to the above embodiments of the present invention. All simple and equivalent changes and modifications made in accordance with the claims and description of this application fall within the protection scope of the claims of this patent. All aspects not described in detail in this invention are conventional technical content.

Claims

1. A method for selective enrichment and rapid detection of IgG glycopeptides in trace samples, characterized in that, Includes the following steps: 1) Selective purification and enzymatic digestion of IgG in trace samples yielded a mixture of polypeptides and glycopeptides; 2) The mixture is subjected to glycopeptide enrichment and desalting using any of the following methods: Option A: A C18 solid-phase extraction column composed of C18 membrane packing and pipette tips is used. After sequential activation with methanol and 70% acetonitrile containing 0.1% hydrofluoric acid, the mixture is loaded and selectively eluted with glycopeptides under low-proportion organic phase conditions to achieve integrated desalting and enrichment. Option B: A solid-phase extraction column consisting of shaped defatted cotton and pipette tips or a 96-well column is used. The mixture is loaded after being activated sequentially with ultrapure water and 80% acetonitrile containing 1% hydrofluoric acid. Then, it is washed with 80% acetonitrile containing 1% hydrofluoric acid to remove salts and hydrophobic peptides. Finally, it is washed with 0.1% hydrofluoric acid aqueous solution to remove glycopeptides, thereby achieving selective enrichment of glycopeptides. 3) The enriched glycopeptides from step 2) were analyzed by rapid liquid chromatography-mass spectrometry, using a chromatographic gradient of less than 5 minutes for separation.

2. The method according to claim 1, characterized in that, In Scheme A, the low-proportion organic phase is 20%-30% acetonitrile containing 0.1% hydrofluoric acid.

3. The method according to claim 1, characterized in that, In Scheme A, the C18 membrane packing material is a single-layer circular C18 membrane packing material sheet with a diameter of 0.8-1.2 mm, and it is filled inside the conical end of a 200 μL low-adsorption pipette tip.

4. The method according to claim 1, characterized in that, In Scheme B, the shaped degreased cotton is a sheet or columnar material with a uniform structure, uniform fiber arrangement, consistent pore size, diameter of 0.8-2.5mm, thickness of 1-5mm, and weight of 0.2-2mg.

5. The method according to claim 1, characterized in that, In the rapid liquid chromatography-mass spectrometry (LC-MS) analysis, the following two secondary mass spectrometry acquisition modes are used: In DDA mode, the acquisition range of full scan mass spectrometry is 700-2000 m / z, and the TopSpeed ​​method is used for MS / MS spectrum acquisition with a cycle time of 2 seconds. In DIA mode, the acquisition range of full scan mass spectrometry is 750-1450 m / z, and MS / MS spectrum acquisition uses a 3 Th isolation window with a maximum injection time of 5 milliseconds; Both DDA and DIA modes use high-energy collision disintegration fragmentation, with the normalized collision energy set to 30%.

6. The method according to claim 1, characterized in that, The micro-clinical sample is 1-5 μL of plasma, tumor interstitial fluid, or cerebrospinal fluid.

7. The method according to claim 1, characterized in that, Step 1) involves the selective purification and enzymatic digestion of IgG, which includes: mixing sample and protein G affinity material at a ratio of 1:2 (V:V), incubating at 800 rpm for 60 minutes at room temperature, washing three times each with 200 μL phosphate buffer and 200 μL ultrapure water, and eluting with 50 μL 0.1% hydrofluoric acid to obtain purified IgG; adding 5 μL of 500 mM tris(hydroxymethyl)aminomethane-hydrochloric acid (pH=8.5) to the purified IgG to adjust the pH to 7.5-8.0, denaturing at 95°C for 10 minutes, and then restoring to room temperature; adding 5 mM tris(2-carboxyethyl)phosphine hydrochloride and reacting at room temperature for 20 minutes, then adding 10 mM indole-3-acetic acid and reacting at room temperature in the dark for 20 minutes; finally, adding trypsin at a mass ratio of enzyme to protein of 1:50 and digesting overnight at 37°C.

8. The method according to claim 1, characterized in that, In Scheme A, after loading the mixture, centrifuge at 500×g for 1 minute; before elution, wash the sample with 100 μL of 0.1% hydrofluoric acid to remove salts, centrifuge at 500×g for 1 minute, and repeat 3 times; the selective elution conditions are: use 100 μL of 20%-30% acetonitrile solution containing 0.1% hydrofluoric acid, centrifuge at 300×g for 1 minute; In Scheme B, after loading the mixture, use 100 μL of 80% acetonitrile containing 1% hydrofluoric acid to remove salts and hydrophobic peptides, centrifuge at 100×g for 30 seconds, repeat 3 times, and finally use 100 μL of 0.1% hydrofluoric acid to elute glycopeptides, centrifuge at 50×g for 30 seconds, and repeat 2 times.

9. The method according to claim 1, characterized in that, In step 3), chromatographic separation was performed on a C18 analytical column. The gradient for rapid liquid chromatography was as follows: mobile phase A was an ultrapure aqueous solution containing 0.1% formic acid, and mobile phase B was a 99.9% acetonitrile solution containing 0.1% formic acid. Initially, phase B was 5% at 0 minutes, linearly increased to 10% at a flow rate of 3 μL / min from 0 to 0.5 minutes, maintained at 10% phase B at a flow rate of 1 μL / min from 0.5 to 1.5 minutes, increased to 16% phase B at a flow rate of 1 μL / min from 1.5 to 3.5 minutes, increased to 30% phase B at a flow rate of 3 μL / min from 3.5 to 4.0 minutes, and washed with 100% phase B at a flow rate of 3 μL / min from 4.0 to 5.0 minutes. The electrospray voltage for mass spectrometry was 1.9 kV, and the capillary temperature was maintained at 320 °C.

10. The application of the method according to any one of claims 1-9 in the discovery of disease biomarkers and high-throughput screening of large clinical cohort samples.

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