CN110130099B - Magnetic nanofiber-based zwitterionic hydrophilic materials for selective capture and recognition of glycopeptides - Google Patents
Magnetic nanofiber-based zwitterionic hydrophilic materials for selective capture and recognition of glycopeptides Download PDFInfo
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Abstract
The invention provides a magnetic nanofiber-based zwitter-ion hydrophilic material for selectively capturing and identifying glycopeptides, which is used for enriching and identifying the glycopeptides; one-dimensional Hydroxyapatite Nanofiber (HN) is used as a carrier to fix Fe3O4Nanoparticles and Au nanoparticles, via the thiol group of the zwitterionic tripeptide L-glutathione with Au and Fe3O4The surface modification is carried out through the affinity interaction between the two to form mHN/Au-GSH nano-fiber. mHN/Au-GSH nanofiber has high glycopeptide detection sensitivity (2fmol), high recovery rate (89.65%), strong binding capacity (100mg/g) and high enrichment selectivity (1: 100).
Description
Technical Field
The invention relates to a magnetic nanofiber-based zwitter-ion hydrophilic material for selectively capturing and identifying glycopeptides, and belongs to the technical field of biochemical analysis.
Background
Proteins/peptides, carbohydrates, nucleic acids, etc. as essential and important biological components mediate most of the fundamental biological processes such as cellular communication, immune reactions and enzymatic reactions. Due to the complex composition and severe interference, the detection and characterization of target biological macromolecules is a great challenge, especially for low abundance analytes. Thus, sample preparation is an indispensable step in the analytical process. The development of affinity materials for selective separation and preconcentration of target biological macromolecules is an effective method to reduce impurity interference and improve detection sensitivity. To date, a variety of materials have been developed, including silica, mesoporous silica, magnetic nanoparticles, Au nanoparticles, polymers, graphene oxide, metal organic frameworks, and porous organic frameworks, for biomacromolecule extraction. The design and synthesis of affinity materials for highly selective capture and identification of target biological macromolecules remains an important research topic.
Glycoproteins are involved in a variety of biological processes and are also biomarkers for several diseases. The comprehensive identification of the glycoprotein not only can understand the physiological process, but also is beneficial to the discovery of biomarkers. The current popular glycopeptide omics analysis method based on mass spectrum comprises proteolysis, glycopeptide enrichment and mass spectrum identification. Based on the structural differences between glycopeptides and non-glycopeptides, several affinity materials including lectins, hydrazine beads, boronic acids, and hydrophilic adsorbents have been introduced to identify and separate glycopeptides and non-glycopeptides abundant in living organisms. In contrast, the hydrophilic interaction separation method has the advantages of high enrichment efficiency, wide glycan specificity, irreversible change of glycan components and the like.
Many hydrophilic materials have been proposed for the enrichment and identification of glycopeptides. Notably, various hydrophilic materials have been combined with magnetic nanoparticles to achieve magnetic response capabilities, making the separation process simple and efficient.
However, the synthesis of these hydrophilic materials often requires complex synthetic procedures, multi-step functionalization, and longer incubation times. This cumbersome process greatly increases the cost of the analytical methods, greatly limiting their application to practical samples. Therefore, more hydrophilic materials with abundant structural features need to be excavated to realize simple and efficient enrichment of glycopeptides.
Disclosure of Invention
The invention aims at the problems, thereby providing a magnetic nanofiber-based zwitter-ion hydrophilic material for selectively capturing and identifying glycopeptides, which has the characteristics of good hydrophilicity, abundant zwitter-ion glutathione molecules and strong magnetic responsiveness, and has better specificity and efficiency in the aspect of glycopeptide enrichment.
The specific technical scheme is as follows:
magnetic nanofiber-based zwitterion hydrophilic material for selectively capturing and identifying glycopeptides, and hydroxyapatite nanofiber is used as a carrier to fix Fe3O4Nanoparticles and Au nanoparticles, via the thiol group of the zwitterionic tripeptide L-glutathione with Au and Fe3O4The surface modification is carried out through the affinity interaction between the two to form mHN/Au-GSH nano-fiber.
Further, the preparation method of the mHN/Au-GSH nano fiber comprises the following steps:
s1: dispersing 2.0g of hydroxyapatite nanofiber and 0.75g of ferric acetylacetonate in 400mL of triethylene glycol to obtain a mixture;
s2: the mixture was transferred to a 1L autoclave and reacted at 220 ℃ for 12 hours, washed with water and ethanol to give fixed Fe3O4A nanofiber product of nanoparticles mHN;
s3: 0.5g of prepared mHN and 25mL of tetrachloroaurate trihydrate were added to 1L of deionized water to obtain a suspension;
s4: the suspension was stirred at 25 ℃ for 12 hours, 50mg of sodium borohydride was added and stirred for 30 minutes to obtain simultaneous fixed Fe3O4Nanofiber product mHN/Au of nanoparticles and Au nanoparticles;
s5: after washing with deionized water, 0.5g of the HN/Au product was dispersed in an aqueous solution containing 2g of glutathione; stirring at 50 deg.C for 10 hr to obtain mHN/Au-GSH nanofiber.
Further, the concentration of tetrachloroauric acid trihydrate in S3 was 2 mg/mL.
Further, mHN/Au-GSH nanofiber is used for enrichment and identification of glycopeptide.
Furthermore, the mHN/Au-GSH nano-fiber can be used for pretreatment before ionization time-of-flight mass spectrometry analysis of human immunoglobulin, chicken avidin protein or bovine serum albumin; the method comprises the following specific steps:
s1: digesting human immunoglobulin, chicken avidin or bovine serum albumin with trypsin;
s2: 400 u g mHN/Au-GSH nanofibers and 2.5 u g human immunoglobulin digests, chicken avidin digests or bovine serum albumin digests into 400 u L buffer solution, at 25 degrees C temperature in 15 minutes;
s3: magnetically separating the material, washing with 2 × 400 μ L buffer solution, and adding 20 μ L eluent to elute and capture glycopeptide;
s4: depositing 1 μ L of the eluate and 1 μ L of DHB on a MALDI plate, and performing MALDI-TOFMS analysis;
in the above pretreatment process, the buffer solution is ACN-H2O-TFA, 92: 7.9: 0.1, v/v/v; the eluent is ACN-H2O, 30: 70, v/v; the concentration of DHB was 25 mg/mL.
Further, the mHN/Au-GSH nanofiber can be used for pretreatment before nano-LC-MS/MS analysis of human serum; the method comprises the following specific steps:
s1: trypsinizing human serum;
s2: 2mg of mHN/Au-GSH nanofibers were incubated with 5. mu.L of human serum digest in 1mL of buffer for 30 min;
s3: adding 2 × 100 μ L of the eluate, and lyophilizing the obtained eluate;
s4: the lyophilized sample was redissolved in 100. mu.L of 25 mmol. multidot.L-1To the sodium bicarbonate solution, PNGaseF was added and incubated overnight at 37 ℃;
s5: freeze-drying the solution again and then carrying out nano-LC-MS/MS analysis;
in the above pretreatment process, the buffer solution is ACN-H2O-TFA, 92: 7.9: 0.1, v/v/v; the eluent is ACN-H2O，30：70，v/v。
The invention has the beneficial effects that:
the mHN/Au-GSH aggregates the one-dimensional structural characteristics of the nanofiber, the excellent hydrophilicity of glutathione and Fe3O4The strong magnetic response capability of the glycopeptide probe is integrated, and the glycopeptide probe has the excellent characteristics of high selectivity, high detection sensitivity, strong binding capability, good recovery efficiency and the like in the aspect of glycopeptide enrichment. Furthermore, the practical potential of mHN/Au-GSH nanofibers for glycoproteome analysis was demonstrated by enrichment of low abundance glycopeptides from human serum. The work provides a way for developing new hydrophilic nano-fibers and application thereof in the biomedical field;
the mHN/Au-GSH nanofiber has high glycopeptide detection sensitivity (2fmol), good recovery rate (89.65%), strong binding capacity (100mg/g) and high enrichment selectivity (1: 100).
Drawings
FIG. 1 is a synthesis process diagram of mHN/Au-GSH nano-fiber.
Fig. 2 is a TEM micrograph of HN (1).
Figure 3 is a TEM micrograph of HN (2).
Fig. 4 is a TEM micrograph (1) of mHN.
Fig. 5 is a TEM micrograph (2) of mHN.
FIG. 6 is a TEM micrograph of mHN/Au nanofibers (1).
FIG. 7 is a TEM micrograph of mHN/Au nanofibers (2).
FIG. 8 is an Energy Dispersive Spectroscopy (EDS) elemental distribution plot of the mHN/Au nanofibers prepared.
FIG. 9 is a graph of Fourier Transform Infrared (FTIR) spectral analysis results; wherein, (i) mHN/Au, (ii) mHN/Au-GSH, and (iii) GSH.
FIG. 10 is a diagram showing the result of XRD analysis; wherein, (i) HN, (ii) mHN, and (iii) mHN/Au.
FIG. 11 is a hysteresis loop curve of mHN and mHN/Au-GSH; wherein, (i) mHN, (ii) mHN/Au-GSH.
FIG. 12 is a MALDI-TOFMS spectrum of a direct analysis of a 0.4pmol human IgG digest.
FIG. 13 is a MALDI-TOFMS spectrum of mHN/Au-GSH enriched nanofibers.
FIG. 14 is a MALDI-TOFMS spectrum of mHN-GSH-enriched nanofibers.
FIG. 15 is a MALDI-TOFMS spectrum of HN/Au-GSH enriched nanofibers.
FIG. 16 is a MALDI-TOFMS spectrum enriched with commercial zwitterionic hydrophilic material.
FIG. 17 is a MALDI-TOFMS spectrum enriched by mHN/Au-GSH nanofibers and deglycosylated by PNGaseF.
FIG. 18 is a MALDI-TOF MS spectrum of a 0.25pmol chicken avidin digest direct analysis.
FIG. 19 is a MALDI-TOFMS spectrum enriched with mHN/Au-GSH nanofibers.
FIG. 20 is a MALDI-TOFMS spectrum of mHN/Au-GSH nanofiber enrichment after digestion of 20mmol human IgG.
FIG. 21 is a MALDI-TOFMS spectrum of mHN/Au-GSH nanofiber enrichment after 2mfmol human IgG digestion.
FIG. 22 is a MALDI-TOFMS spectrum of 20fmol human IgG after digestion and enrichment with commercial zwitterionic hydrophilic material.
FIG. 23 is a MALDI-TOFMS spectrum of 2fmol human IgG after digestion and enrichment with commercial zwitterionic hydrophilic material.
FIG. 24 is a MALDI-TOFMS spectrum of a peptide mixture containing a digest of human IgG and a digest of BSA analyzed directly.
FIG. 25 is a MALDI-TOFMS spectrum of a peptide mixture containing a digest of human IgG and a digest of BSA after enrichment with mHN/Au-GSH nanofibers.
Fig. 26 is a graph in which 1: MALDI-TOFMS spectrum of peptide mixture containing human IgG digest and BSA digest after enrichment of Au-GSH nanofibers at a mass ratio of mHN/100. In fig. 12-26, the glycopeptide peaks are labeled with the symbol "×".
Detailed Description
In order to make the technical solution of the present invention clearer and more clear, the present invention is further described below, and any solution obtained by substituting technical features of the technical solution of the present invention with equivalents and performing conventional reasoning falls within the scope of the present invention.
A magnetic nanofiber-based zwitterion hydrophilic material for selectively capturing and identifying glycopeptides utilizes hydroxyapatite nanofibers as a carrier to fix Fe3O4Nanoparticles and Au nanoparticles, via the thiol group of the zwitterionic tripeptide L-glutathione with Au and Fe3O4The surface modification is carried out through the affinity interaction between the two to form mHN/Au-GSH nano-fiber.
Further, the preparation method of the mHN/Au-GSH nano fiber comprises the following steps:
s1: dispersing 2.0g of hydroxyapatite nanofiber and 0.75g of ferric acetylacetonate in 400mL of triethylene glycol to obtain a mixture;
s2: the mixture was transferred to a 1L autoclave and reacted at 220 ℃ for 12 hours, washed with water and ethanol to give fixed Fe3O4A nanofiber product of nanoparticles mHN;
s3: 0.5g of prepared mHN and 25mL of tetrachloroauric acid trihydrate were added to 1L of deionized water to obtain a suspension;
s4: the suspension was stirred at 25 ℃ for 12 hours, 50mg of sodium borohydride was added and stirred for 30 minutes to obtain simultaneous fixed Fe3O4Nanofiber product mHN/Au of nanoparticles and Au nanoparticles;
s5: after washing with deionized water, 0.5g of the HN/Au product was dispersed in an aqueous solution containing 2g of glutathione; stirring at 50 deg.C for 10 hr to obtain mHN/Au-GSH nanofiber.
Further, the concentration of tetrachloroauric acid trihydrate in S3 was 2 mg/mL.
Further, mHN/Au-GSH nanofiber is used for enrichment and identification of glycopeptide.
Furthermore, the HN/Au-GSH nano-fiber can be used for pretreatment before ionization time-of-flight mass spectrometry analysis of human immunoglobulin, chicken avidin protein or bovine serum albumin; the method comprises the following specific steps:
s1: digesting human immunoglobulin, chicken avidin or bovine serum albumin with trypsin;
s2: 400 u g mHN/Au-GSH nanofibers and 2.5 u g human immunoglobulin digests, chicken avidin digests or bovine serum albumin digests into 400 u L buffer solution, at 25 degrees C temperature in 15 minutes;
s3: magnetically separating the material, washing with 2 × 400 μ L buffer solution, and adding 20 μ L eluent to elute and capture glycopeptide;
s4: depositing 1 μ L of the eluate and 1 μ L of DHB on a MALDI plate, and performing MALDI-TOFMS analysis;
in the above pretreatment process, the buffer solution is ACN-H2O-TFA, 92: 7.9: 0.1, v/v/v; eluentIs ACN-H2O, 30: 70, v/v; the concentration of DHB was 25 mg/mL.
Further, the mHN/Au-GSH nanofiber can be used for pretreatment before nano-LC-MS/MS analysis of human serum; the method comprises the following specific steps:
s1: trypsinizing human serum;
s2: 2mg of mHN/Au-GSH nanofibers were incubated with 5. mu.L of human serum digest in 1mL of buffer for 30 min;
s3: adding 2 × 100 μ L of the eluate, and lyophilizing the obtained eluate;
s4: the lyophilized sample was redissolved in 100. mu.L of 25 mmol. multidot.L-1To the sodium bicarbonate solution, PNGaseF was added and incubated overnight at 37 ℃;
s5: freeze-drying the solution again and then carrying out nano-LC-MS/MS analysis;
in the above pretreatment process, the buffer solution is ACN-H2O-TFA, 92: 7.9: 0.1, v/v/v; the eluent is ACN-H2O，30：70，v/v。
For further understanding of the present invention, the following embodiments are provided to illustrate the technical solutions of the present invention in detail, and the scope of the present invention is not limited by the following embodiments.
mHN/characterization of Au-GSH nanofibers
The synthesis of magnetic nanofiber-based zwitterionic hydrophilic materials (mHN/Au-GSH nanofibers) is given in FIG. 1. Briefly, one-dimensional Hydroxyapatite Nanofiber (HN) is synthesized by a solvothermal reaction method and used as modified Fe3O4Nanoparticles and Au nanoparticles. Subsequently, sulfhydryl groups in Glutathione (GSH) were reacted with Au and Fe3O4The nanoparticles react to allow glutathione to be immobilized on the magnetic nanofibers. The mHN/Au-GSH nanofiber has the characteristics of nanofiber, magnetic response capability and hydrophilic glutathione group, and can be used as an adsorbent to specifically capture glycopeptides in the sample preparation process of mass spectrometry glycopeptide omics research.
FIGS. 2, 3, 4, 5, 6 and 7 show the shapes of HN, mHN and mHN/Au nanofibers producedState. HN consists of hydroxyapatite nanowires with pronounced one-dimensional features, including nanometer-sized diameters of 10-20nm, high aspect ratio, and self-assembly into nanofibers (fig. 2, fig. 3). Compared to the smooth surface of HN, a large number of small nanoparticles with an average diameter of about 12nm were decorated at mHN (fig. 4, fig. 5). In addition, after the Au nanoparticles are fixed on mHN, more small nanoparticles appear on the MHN surface, and mHN/Au nanofibers are formed. The average particle size of the modified gold nanoparticles was calculated to be about 11.6nm using the Scherrer equation according to the X-ray diffraction (XRD) analysis results. In addition, the spatial element distribution of P, Ca, Fe and Au from mHN/Au nanofibers was studied using Energy Dispersive Spectroscopy (EDS) elemental mapping. As shown in fig. 8, the four element signals are uniformly localized, further confirming the signal peaks, revealing Fe3O4And successful immobilization of Au nanoparticles in mHN/Au nanofibers.
Modification of glutathione was confirmed by Fourier Transform Infrared (FTIR) spectroscopic analysis. In FIG. 9, a hydroxyl group (3573 cm)-1)，PO4 3-Radicals (1093, 1031, 962, 605 and 565cm-1) Absorption peaks of (2), adsorbed water was observed in the infrared spectrum of mHN/Au (3436 and 1633 cm)-1)。565cm-1Absorption peak and Fe3O4(560cm-1) Overlap of characteristic absorption peaks of (a). In addition, fingerprint peaks belonging to glutathione were clearly found in fig. 9. These results demonstrate successful glutathione immobilization in Fe3O4And Au nanofibers.
In addition, the structure was also analyzed by XRD. In fig. 10, the characteristic peak of HN is consistent with the analysis result of hydroxyapatite. In contrast, the relative increase in intensity of the diffraction peak marked with the black symbol' at 35.5 ° is due to Fe in the mHNX ray diffraction pattern3O4Due to the magnetic component of (a). In addition, in the XRD pattern of mHN/Au nanofiber, a new peak with black symbol '#' at 38.3 ℃ belongs to Au.
As shown in FIG. 11, hysteresis loops of mHN and mHN/Au-GSH were determined, indicating that these two materials have a supermagnetic characteristic. The saturation magnetization values of mHN and mHN/Au-GSH were found to be 6, respectively.76 and 5.51emu g-1. Although the final nanofibers have a relatively low saturation magnetization value, it is sufficient to allow rapid magnetic separation of the nanofibers from aqueous solutions.
Glycopeptide enrichment performance of mHN/Au-GSH nanofiber measured by standard glycoprotein digestion method
Human immunoglobulin IgG trypsin digest was used as a model sample to study the glycopeptide enrichment performance of the prepared mHN/Au-GSH nanofibers. As shown in fig. 12, no glycopeptide peaks were observed in the non-enriched human IgG digest. However, after enrichment with mHN/Au-GSH nanofibers, the glycopeptide peak with strong signal intensity dominates the mass spectrum and there is hardly any interference from non-glycopeptide peaks (FIG. 13). We performed a total of 21 glycopeptides for detection and identification. In addition, mHN-GSH nanofibers, non-magnetic nanofibers (HN/Au-GSH), and commercial ZIC-HILIC materials were used as controls. In fig. 14, 15 and 16, only 17, 20 and 18 glycopeptide peaks were detected, respectively. After mHN/Au-GSH nanofiber enrichment, the identified glycopeptide peak has signal intensity and signal-to-noise ratio (S/N) which are obviously higher than those of a control material after treatment. For example, glycopeptide peaks enriched with mHN/Au-GSH, mHN-GSH, HN/Au-GSH, and commercial ZIC-HILIC materials have S/N ratios of 810.7, 464.7, 564.5, and 492.6 at a mass to charge ratio of 2764.1, respectively. The result shows that the enrichment performance of mHN/Au-GSH is superior to that of mHN-GSH, HN/Au-GSH and commercial ZIC-HILIC materials. In addition, the captured glycopeptide is deglycosylated by PNGaseF (glycoamidase). In FIG. 17, all the glycopeptide peaks detected disappeared, leaving only the two major deamidated peptides (EEQFN # STFR, EEQYN # STYR), indicating that all the peaks observed in FIG. 13 are related to N-bond glycopeptides. The unique one-dimensional structure, abundant zwitterion groups and strong magnetic responsiveness contribute to providing excellent enrichment performance. These results all confirm that the prepared mHN/Au-GSH nanofiber has high enrichment specificity and efficiency on glycopeptide.
In addition, a trypsin digest of chicken avidin was also used. Mass spectra of chicken avidin digests As shown in FIG. 18, 8 glycopeptide peaks with low signal intensity and S/N ratio were observed due to the simultaneous presence of a large number of non-glycopeptides. Importantly, after the selective enrichment process using mHN/Au-GSH nanofibers, the 14 glycopeptide peaks in the mass spectrum (FIG. 19) were affected with improved signal intensity and S/N ratio. These analysis results demonstrate that the prepared mHN/Au-GSH nanofibers have high selectivity and efficiency for general glycopeptide enrichment.
The detection sensitivity of the prepared mHN/Au-GSH nanofiber to glycopeptide is determined. As shown in fig. 20, 12 glycopeptide peaks were clearly observed in a 20fmol human immunoglobulin IgG digest after the enrichment process. When the amount of human IgG digestion was as low as 2fmol (FIG. 21), four glycopeptide peaks were still observed, with a maximum S/N ratio of 22.8. The detection sensitivity of mHN/Au-GSH nanofiber for glycopeptide was considered to be 2 fmol. For comparison, the detection sensitivity of commercial hydrophilic materials was also tested. When the amount of human IgG digestion was 20fmol and enriched with commercial hydrophilic material, only 8 glycopeptide peaks could be detected (fig. 22). On the other hand, when the amount of human IgG digested was only 2fmol (FIG. 23), no glycopeptide peak was observed. The mHN/Au-GSH nanofiber studied at this time has higher detection sensitivity on abundant low-abundance glycopeptides, and the performance of the nanofiber is superior to that of CS @ PGMA @ IDA-Ti4+(45fmol)、GO-Fe3O4/SiO2AuNWs/L-cysteine (10fmol), MoS2Au-NP-L-cysteine (10fmol) and Fe3O4@ TpPa are several hydrophilic materials.
According to the previous literature, the recovery of enrichment of 6 targeted glycopeptides released from human IgG was evaluated by isotopic dimethyl labelling. The average enrichment recovery achieved was 89.65%. Compared with Resin @ Polysaccharide (73%), GO/Fe which are reported in the past3O4/Au/PEG(59.2％)、Fe3O4@MPS@PMAC(82％)、MIL-101(NH2) Compared with several hydrophilic materials of @ Au-Cysteine (80%) and Resin/Au-Cysteine (79%), the mHN/Au-GSH nanofiber prepared by the method has better capture capability.
At the same time, the binding capacity of the confirmed mHN/Au-GSH nanofiber is measured. A fixed mass of human IgG digest (3. mu.g) was incubated with a mass of mHN/Au-GSH. After a period of time, the detection is carried out when mHN/Au-GSH is usedAt 30. mu.g, the signal intensities of the six selected glycopeptides reached a maximum. mHN/Au-GSH nano-fiber with glycopeptide binding capacity of 100mg g-1. This is probably due to the strong multivalent hydrophilic interaction between the glycopeptide and the MHN/Au-GSH nanofibers due to the abundance of zwitterionic hydrophilic groups. The experiment result shows that the combination capability of the MHN/Au-GSH nano-fiber is stronger than that of Fe3O4@CSMCNCs(17.5mg/g)、Fe3O4-DA-Maltose (43mg/g) and Fe3O4@PGMA@Au-L-cysteine(75mg/g)。
The enrichment selectivity of mHN/Au-GSH nanofibers was further investigated. As shown in fig. 24, by directly analyzing the polypeptide mixture containing the human IgG digest and the BSA digest, we found that the non-glycopeptide peak was dominant in the mass spectrum and that the glycopeptide peak was not clearly identified. However, after enrichment with mHN/Au-GSH nanofibers, the non-glycopeptides disappeared significantly, and the signal intensities of 19 and 17 glycopeptides detected were significantly improved (FIGS. 25 and 26), which provides more evidence for enrichment of glycopeptides with mHN/Au-GSH nanofibers.
Selective enrichment and identification of glycopeptides in human serum
Human serum has attracted considerable attention as a typical clinical specimen, a potential biomarker for diagnosing diseases. The advantages of mHN/Au-GSH nanofiber in glycopeptide enrichment prove that human serum glycoprotein is analyzed according to the scheme of proteolysis, mHN/Au-GSH nanofiber glycopeptide enrichment, PNGase F deglycosylation and mass spectrum identification. As a result, a total of 246 glycopeptides of 104 glycoproteins were identified by only three independent analyses of 1. mu.L human serum. In contrast, commercial zwitterionic hydrophilic materials identified only 162 glycopeptides from 69 glycoproteins. The structural differences of these affinity materials contribute to their diverse abilities in glycopeptide enrichment. These results indicate that the prepared mHN/Au-GSH nanofiber has high specificity and high efficiency in capturing low-abundance glycopeptides, and has great potential in researching the glycoproteome of complex biological samples.
The experimental procedures in this example are conventional, unless otherwise specified. The experimental materials used in the present examples were all purchased from the market unless otherwise specified.
Transmission Electron Microscope (TEM) images and Energy Dispersive Spectroscopy (EDS) elemental mapping were obtained with a transmission electron microscope (feitecnag 2F20, USA). The infrared spectra were obtained using a Thermo Nicolet380 Fourier transform infrared spectrometer (Nicolet, Wisconsin, USA). X-ray diffractometry (RigakuD/max2550V, CuK alpha radiation,
) An X-ray powder diffraction (XRD) pattern was recorded. The saturation magnetization curve was tested on a physical property measurement system (PPMS, USA) at room temperature. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) experiments were performed in linear positive mode on an AB Sciex5800 mass spectrometer (ABSciex, CA) using a Nd/YAG laser at 355 nm.
Claims (2)
1. The magnetic nanofiber-based zwitterion hydrophilic material for selectively capturing and identifying glycopeptides is characterized in that the magnetic nanofiber-based zwitterion hydrophilic material is prepared by fixing Fe by taking hydroxyapatite nanofiber as a carrier3O4Nanoparticles and Au nanoparticles, via the thiol group of the zwitterionic tripeptide L-glutathione with Au and Fe3O4The surface modification is carried out by the affinity interaction, and the specific preparation method comprises the following steps:
s1: dispersing 2.0g of hydroxyapatite nanofiber and 0.75g of ferric acetylacetonate in 400mL of triethylene glycol to obtain a mixture;
s2: the mixture was transferred to a 1L autoclave and reacted at 220 ℃ for 12 hours, washed with water and ethanol to give fixed Fe3O4A nanofiber product of nanoparticles mHN;
s3: 0.5g of prepared mHN and 25mL of tetrachloroauric acid trihydrate were added to 1L of deionized water to obtain a suspension;
s4: the suspension was stirred at 25 ℃ for 12 hours, 50mg of sodium borohydride was added and stirred for 30 minutes to obtain simultaneous fixed Fe3O4Nanofiber product mH of nanoparticles and Au nanoparticlesN/Au；
S5: after washing with deionized water, 0.5g of the mHN/Au product was dispersed in an aqueous solution containing 2g of the zwitterionic tripeptide L-glutathione; stirring for 10 hours at 50 ℃ to obtain the magnetic nanofiber-based zwitterionic hydrophilic material.
2. The magnetic nanofiber-based zwitterionic hydrophilic material for selectively capturing and recognizing a glycopeptide according to claim 1, wherein the concentration of tetrachloroaurate trihydrate in S3 is 2 mg/mL.
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