Synchronous enrichment and identification method of N-linked glycopeptide and O-linked glycopeptide

Abstract

The invention discloses a method for synchronously enriching and identifying N-linked glycopeptides and O-linked glycopeptides, and relates to the technical field of material analysis chemistry and organic chemistry, the method is characterized in that glycopeptides are enriched by adopting a column solid-phase extraction mode or a dispersive solid-phase extraction mode, and a glycopeptide sample is subjected to mass spectrometry. The glycopeptide enrichment material is a material obtained by carrying out copolymerization reaction on different types of hydrophilic monomer molecules with acrylic acid groups on the surface of porous silica gel by utilizing a reversible-fracture-chain transfer free radical polymerization reaction mechanism initiated by a matrix surface, and can be used for glycopeptide enrichment. According to the method, by controlling conditions such as pH and organic phase concentration in the enrichment process, step-by-step enrichment of N-linked glycopeptide and O-linked glycopeptide can be realized, high-selectivity enrichment of two glycopeptides in a complex mixture (high non-glycopeptide doping ratio) can be realized, and the capacity of identifying glycosylation in glycoprotein is remarkably improved.

Description

Technical Field

[0001] The invention relates to the technical field of material analytical chemistry and organic chemistry, and in particular to a method for synchronous enrichment and identification of N-linked glycopeptides and O-linked glycopeptides. Background Art

[0002] Protein glycosylation modification plays an important regulatory role in the operation of biological cells. Therefore, studying protein glycosylation at the molecular level is of great significance to revealing its regulatory mechanism in life processes.

[0003] Protein glycosylation is divided into N-glycosylation modification and O-glycosylation modification. The research on N-glycosylation modification alone is relatively mature, and the enrichment methods and identification strategies are also relatively rich. However, O-glycosylation modification is more difficult to study than N-glycosylation modification, because O-sugar chains are usually shorter in length and have complex core structures. O-linked glycopeptides and N-linked glycopeptides usually exist simultaneously in enzymatically hydrolyzed biological samples. Therefore, the enrichment and identification of O-linked glycopeptides is very challenging.

[0004] The existing methods for enriching O-linked glycopeptides mainly include lectin method, hydrazine chemistry method and hydrophilic interaction chromatography, etc. However, these methods have certain limitations: lectin affinity chromatography can only use fixed lectins to enrich glycopeptides or glycoproteins, so there is a problem of low glycosylation coverage; although hydrazine chemistry has high selectivity for glycopeptides, it is easy to destroy sugar chain information during the elution process.

[0005] Therefore, the prior art still has the technical problem of being unable to selectively enrich and identify low-content N-linked glycopeptides and O-linked glycopeptides in biological samples quickly and effectively. Summary of the invention

[0006] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for the simultaneous enrichment and identification of N-linked glycopeptides and O-linked glycopeptides. By providing a method for the simultaneous enrichment and identification of N-linked glycopeptides and O-linked glycopeptides with high selectivity, simple operation and good repeatability, low-content N-linked glycopeptides and O-linked glycopeptides in biological samples can be selectively enriched and identified efficiently.

[0007] The first aspect of the present invention is to provide a method for the simultaneous enrichment of N-linked glycopeptides and O-linked glycopeptides, characterized in that a glycopeptide enrichment material is contacted with a glycoprotein hydrolysate, a column solid phase extraction mode or a dispersed solid phase extraction mode is used to separate and enrich the glycopeptides, and the glycopeptide sample is subjected to mass spectrometry analysis, wherein the molecular structure of the glycopeptide enrichment material is:

[0008]

[0009] The glycopeptide enrichment material is generated by a reversible-fragmentation-chain transfer free radical polymerization mechanism, wherein a hydrophilic monomer molecule with an acrylic acid group is subjected to a surface-initiated copolymerization reaction on the surface of a porous silica gel, wherein X represents a hydrophilic monomer molecule, and R represents a chain transfer agent group. The particle size of the porous silica gel is 0.2 μm-20 μm, and the pore size is

[0010] According to one aspect of the above technical solution, the step of separating and enriching glycopeptides using a column solid phase extraction mode comprises:

[0011] 1) loading the glycopeptide enrichment material into the tip of a pipette or a gel loading tip to form a micro solid phase extraction column, activating and balancing the micro solid phase extraction column with an activation solution and a balancing solution, dissolving the protein hydrolysate in the balancing solution, and transferring the glycopeptide enrichment material to the protein hydrolysate in a mass ratio of 100:1 to 1000:1;

[0012] 2) washing the micro solid phase extraction column with an organic solution having a pH value of 1-7 and a volume of 5-200 times the column volume;

[0013] 3) Using 5-100 column volumes and an eluent with a pH value of 1-12 for elution to obtain glycopeptides.

[0014] According to one aspect of the above technical solution, the step of separating and enriching glycopeptides using a dispersed solid phase extraction mode comprises:

[0015] 1) activating the glycopeptide enriched material with an activation solution, mixing the glycopeptide enriched material with a protease hydrolysate at a mass ratio of 100:1-1000:1, incubating for 0.5 minutes to 12 hours, separating by filtration or centrifugation, discarding the supernatant, and collecting the precipitate;

[0016] 2) washing the precipitate with an organic solution having a pH value of 1-7;

[0017] 3) The washed precipitate is eluted with an eluent having a pH value of 1-12, separated by filtration or centrifugation, and the supernatant is collected to obtain the glycopeptide.

[0018] According to one aspect of the above technical solution, the organic solution, the activation solution and the balancing solution are all mixed solutions of organic solvent, organic acid and water, and the eluent contains an alkaline solvent.

[0019] According to one aspect of the above technical solution, the organic solvent is acetonitrile, methanol or ethanol, the volume concentration of the organic solvent is 50%-95%, and the organic acid is formic acid, acetic acid or trifluoroacetic acid, the volume concentration of the organic acid is 0.1-5%.

[0020] According to one aspect of the above technical solution, the volume concentration of the organic solvent in the activation solution is 10%-50%, and the pH value is 1-7.

[0021] According to one aspect of the above technical solution, the volume concentration of the organic solvent in the equilibrium solution is 50%-95%, and the pH value is 1-7.

[0022] According to one aspect of the above technical solution, the volume concentration of the alkaline solvent in the eluent is 0.1%-30%, and the pH value is 8-12.

[0023] According to one aspect of the above technical solution, the volume concentration of the alkaline solvent in the eluent is 0.1%-5%, and the pH value is 1-7.

[0024] The second aspect of the present invention is to provide a method for simultaneously identifying N-linked glycopeptides and O-linked glycopeptides, which is used to identify the glycopeptide enriched material obtained by simultaneously enriching N-linked glycopeptides and O-linked glycopeptides in the method shown in the above technical scheme, and the method comprises:

[0025] Using glycoproteomics retrieval software, by adding detailed information of N-glycan and O-glycan sugar libraries in the sugar library at the same time, setting the enzyme cleavage method and modification type that match the experimental conditions, and then using simultaneous retrieval to simultaneously identify N-linked glycopeptides and O-linked glycopeptides.

[0026] Compared with the prior art, the method for simultaneous enrichment and identification of N-linked glycopeptides and O-linked glycopeptides shown in the present invention has the following beneficial effects:

[0027] The present invention brings a glycopeptide enrichment material into contact with a glycoprotein hydrolysate, separates and enriches glycopeptides by adopting a column solid phase extraction mode or a dispersed solid phase extraction mode, and performs mass spectrometry analysis on the glycopeptide sample. The glycopeptide enrichment material is generated by a reversible-fragmentation-chain transfer free radical polymerization reaction mechanism, specifically, a hydrophilic monomer molecule with an acrylic acid group is subjected to a surface-initiated copolymerization reaction on the surface of a porous silica gel, and exhibits a high selectivity characteristic when simultaneously enriching glycopeptides, and can achieve efficient simultaneous enrichment of N-linked glycopeptides and O-linked glycopeptides; and the glycopeptide enrichment material can be conveniently loaded into extraction columns of different lengths and different inner diameters, and can also be directly added to a centrifuge tube, and is simple to operate, easy to repeat, and particularly suitable for separation and enrichment of glycopeptides in trace biological samples; and the glycopeptide enrichment material can be directly used for electrospray ionization-mass spectrometry (ESI-MS) or matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALD-TOF-MS), thereby improving the detection limit and sensitivity of the mass spectrometry. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0029] Figure 1 A molecular schematic diagram of a polymer material for simultaneous enrichment of N-linked glycopeptides and O-linked glycopeptides prepared by reversible-fragmentation-chain transfer free radical polymerization using hydrophilic serine and arginine as monomers;

[0030] Figure 2 Schematic diagram of electron microscopy characterization (A) and infrared characterization (B) of the synchronous enrichment polymer material prepared using hydrophilic serine and arginine as monomers;

[0031] Figure 3 Schematic diagram of the recovery rate of N-linked glycopeptides and O-linked glycopeptides of the simultaneous enrichment polymer material prepared with hydrophilic serine and arginine as monomers using standard protein fetuin enzymatic hydrolysate. DETAILED DESCRIPTION

[0032] In order to make the purpose, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the accompanying drawings. Several embodiments of the present invention are shown in the accompanying drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0034] The first aspect of the present invention is to provide a method for the simultaneous enrichment of N-linked glycopeptides and O-linked glycopeptides, wherein a glycopeptide enrichment material is contacted with a glycoprotein hydrolysate, a column solid phase extraction mode or a dispersed solid phase extraction mode is used to separate and enrich the glycopeptides, and the glycopeptide sample is subjected to mass spectrometry analysis, wherein the molecular structure of the glycopeptide enrichment material is:

[0035]

[0036] The glycopeptide enrichment material is generated by a reversible-fragmentation-chain transfer free radical polymerization mechanism, wherein a hydrophilic monomer molecule with an acrylic acid group is subjected to a surface-initiated copolymerization reaction on the surface of a porous silica gel, wherein X represents a hydrophilic monomer molecule, and R represents a chain transfer agent group. The particle size of the porous silica gel is 0.2 μm-20 μm, and the pore size is

[0037] Furthermore, the step of separating and enriching glycopeptides using a column solid phase extraction mode comprises:

[0038] 1) loading the glycopeptide enrichment material into the tip of a pipette or a gel loading tip to form a micro solid phase extraction column, activating and balancing the micro solid phase extraction column with an activation solution and a balancing solution, dissolving the protein hydrolysate in the balancing solution, and transferring the glycopeptide enrichment material to the protein hydrolysate in a mass ratio of 100:1 to 1000:1;

[0039] 2) washing the micro solid phase extraction column with an organic solution having a pH value of 1-7 and a volume of 5-200 times the column volume;

[0040] 3) Using 5-100 column volumes and an eluent with a pH value of 1-12 for elution to obtain glycopeptides.

[0041] Furthermore, the step of separating and enriching glycopeptides using a dispersed solid phase extraction mode comprises:

[0042] 1) activating the glycopeptide enriched material with an activation solution, mixing the glycopeptide enriched material with a protease hydrolysate at a mass ratio of 100:1-1000:1, incubating for 0.5 minutes to 12 hours, separating by filtration or centrifugation, discarding the supernatant, and collecting the precipitate;

[0043] 2) washing the precipitate with an organic solution having a pH value of 1-7;

[0044] 3) The washed precipitate is eluted with an eluent having a pH value of 1-12, separated by filtration or centrifugation, and the supernatant is collected to obtain the glycopeptide.

[0045] Furthermore, the organic solution, the activation solution and the balancing solution are all mixed solutions of organic solvent, organic acid and water, and the eluent contains an alkaline solvent.

[0046] Furthermore, the organic solvent is acetonitrile, methanol or ethanol, the volume concentration of the organic solvent is 50%-95%, the organic acid is formic acid, acetic acid or trifluoroacetic acid, the volume concentration of the organic acid is 0.1-5%.

[0047] Furthermore, the volume concentration of the organic solvent in the activation solution is 10%-50%, and the pH value is 1-7.

[0048] Furthermore, the volume concentration of the organic solvent in the equilibrium solution is 50%-95%, and the pH value is 1-7.

[0049] Furthermore, the volume concentration of the alkaline solvent in the eluent is 0.1%-30%, and the pH value is 8-12.

[0050] Furthermore, the volume concentration of the alkaline solvent in the eluent is 0.1%-5%, and the pH value is 1-7.

[0051] The second aspect of the present invention is to provide a method for simultaneously identifying N-linked glycopeptides and O-linked glycopeptides, which is used to identify the glycopeptide enriched material obtained by simultaneously enriching N-linked glycopeptides and O-linked glycopeptides in the method described in the above technical scheme, and the method comprises:

[0052] Using glycoproteomics retrieval software, by adding detailed information of N-glycan and O-glycan sugar libraries in the sugar library at the same time, setting the enzyme cleavage method and modification type that match the experimental conditions, and then using simultaneous retrieval to simultaneously identify N-linked glycopeptides and O-linked glycopeptides.

[0053] Compared with the prior art, the method for simultaneous enrichment and identification of N-linked glycopeptides and O-linked glycopeptides shown in the present invention has the following beneficial effects:

[0054] The present invention brings a glycopeptide enrichment material into contact with a glycoprotein hydrolysate, separates and enriches glycopeptides by adopting a column solid phase extraction mode or a dispersed solid phase extraction mode, and performs mass spectrometry analysis on the glycopeptide sample. The glycopeptide enrichment material is generated by a reversible-fragmentation-chain transfer free radical polymerization reaction mechanism, specifically, a hydrophilic monomer molecule with an acrylic acid group is subjected to a surface-initiated copolymerization reaction on the surface of a porous silica gel, and exhibits a high selectivity characteristic when simultaneously enriching glycopeptides, and can achieve efficient simultaneous enrichment of N-linked glycopeptides and O-linked glycopeptides; and the glycopeptide enrichment material can be conveniently loaded into extraction columns of different lengths and different inner diameters, and can also be directly added to a centrifuge tube, and is simple to operate, easy to repeat, and particularly suitable for separation and enrichment of glycopeptides in trace biological samples; and the glycopeptide enrichment material can be directly used for electrospray ionization-mass spectrometry (ESI-MS) or matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALD-TOF-MS), thereby improving the detection limit and sensitivity of the mass spectrometry.

[0055] Embodiment 1

[0056] like Figure 1 As shown, Example 1 of the present invention provides a method for simultaneous enrichment of N-linked glycopeptides and O-linked glycopeptides, N-linked glycopeptides are N-glycopeptides, and O-linked glycopeptides are O-glycopeptides. The simultaneous enrichment method shown in this embodiment includes:

[0057] First, RAFT reagent 4-cyanovaleric acid dithiobenzoate (CPADB) (0.204 g, 0.73 mmol), N-hydroxysuccinimide (NHS) (0.11 g, 0.949 mmol), and 1,3-dicyclohexylcarbodiimide (DCC) (0.151 g, 0.73 mmol) were added into a flask and mixed, nitrogen was introduced, 5 mL of anhydrous dichloromethane and 100 mL of triethylamine (TEA) were added and mixed again, and the mixture was reacted at room temperature in a nitrogen atmosphere and protected from light for 48 hours to obtain NH2-CPADB.

[0058] Then, amino silica (NH2-SiO2) microspheres were added to the flask, nitrogen was introduced, and N H2 -CPADB and 8 mL tetrahydrofuran (THF) were reacted at room temperature in a nitrogen atmosphere and protected from light for 48 hours to obtain SiO2-CPADB.

[0059] Then, acrylic acid-modified serine (Ser)-arginine (Arg) monomer (AA-SR) (52.6 mg, 180 mmol), SiO2-CPADB (50 mg, 15 mmol), 1 mL of ethanol, and 1 mL of sodium acetate solution (7.2 mM) were added to the flask and mixed. Three freeze-pump-thaw cycles were performed to remove oxygen from the flask. AIBA (4.9 mg, 18 mmol) was slowly added and the reaction was carried out at 55 °C for 48 hours.

[0060] Then, the obtained material was dispersed in a flask with 1 mL of water, 20 μL of hydrogen peroxide was added, and the mixture was reacted at 70° C. for 4 hours to obtain SR-SiO 2 .

[0061] Finally, the obtained SR-SiO2 was washed three times with water, then centrifuged at 5000 rpm for 3 min and dried before storage.

[0062] Embodiment 2

[0063] The N-linked glycopeptide and O-linked glycopeptide simultaneous enrichment polymer material prepared by reversible-fragmentation-chain transfer free radical polymerization reaction using hydrophilic serine (Ser)-arginine (Arg) as monomers (AA-SR) was characterized by electron microscopy and infrared. Figure 2 As shown in Figure A, by comparing with silica gel (Figure A, top), it can be found that after the polymerization reaction, the polymerization reaction was initiated on the silica gel surface and a polymer was generated (Figure A, bottom); in the infrared characterization results of Figure B, the characteristic shifts of 1690 and 1590 cm -1 It was proved that the reversible-fragmentation-chain transfer free radical polymerization reaction occurred successfully, and the hydrophilic serine and arginine were modified on the surface of the material.

[0064] Embodiment 3

[0065] The recovery rate of N-linked glycopeptides and O-linked glycopeptides in standard protease hydrolysate was determined using a polymer material based on hydrophilic serine (Ser)-arginine (Arg) monomers (AA-SR) and prepared by reversible-fragmentation-chain transfer free radical polymerization. Figure 3 As shown, two N-linked glycopeptides and two O-linked glycopeptides were selected as references, and the recovery rates of N-linked glycopeptides and O-linked glycopeptides were calculated. The recovery rates were all above 90%, proving the feasibility of the simultaneous enrichment and identification method of N-linked glycopeptides and O-linked glycopeptides.

[0066] Embodiment 4

[0067] The N-linked glycopeptide and O-linked glycopeptide enrichment material was prepared according to the method described in Example 1, and the N-linked glycopeptide and O-linked glycopeptide were enriched in a centrifugal operation mode.

[0068] 1mg glycopeptide enrichment material is loaded into a centrifuge tube, bovine fetal protein hydrolysate and bovine serum albumin protein hydrolysate are mixed according to the mass molar ratio of 1:1000 and added into a centrifuge tube (wherein the bovine fetal protein hydrolysate mass is 5mg), hatched for 30min, supernatant is collected after centrifugation, precipitate is hatched for 5min using 100mL of an aqueous solution containing 80% acetonitrile / volume concentration 0.1% formic acid (pH=3) by volume concentration, supernatant is collected after centrifugation, precipitate is hatched for 5min using 100mL of an aqueous solution containing 75% acetonitrile / volume concentration 0.1% formic acid (pH=3) by volume concentration, supernatant is collected after centrifugation, precipitate is eluted using 30mL of 10% (v / v) ammonia solution (pH=12). After the eluent is spin-dried, it is analyzed on a mass spectrometer. Wherein, the above-mentioned whole process is carried out under 20°C conditions.

[0069] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0070] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for those of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for simultaneous enrichment of N-linked glycopeptides and O-linked glycopeptides, characterized in that: The glycopeptide enrichment material is contacted with the glycoprotein enzymatic hydrolysate, the glycopeptide is separated and enriched by using a column solid phase extraction mode or a dispersed solid phase extraction mode, and the glycopeptide sample is subjected to mass spectrometry analysis. The molecular structure of the glycopeptide enrichment material is: The glycopeptide enrichment material is generated by a reversible-fragmentation-chain transfer free radical polymerization mechanism, wherein a hydrophilic monomer molecule with an acrylic acid group is subjected to a surface-initiated copolymerization reaction on the surface of a porous silica gel, wherein X represents a hydrophilic monomer molecule, and R represents a chain transfer agent group. The particle size of the porous silica gel is 0.2 μm-20 μm, and the pore size is 2. The method for simultaneous enrichment of N-linked glycopeptides and O-linked glycopeptides according to claim 1, characterized in that: The steps of separating and enriching glycopeptides using column solid phase extraction mode include: 1) loading the glycopeptide enrichment material into the tip of a pipette or a gel loading tip to form a micro solid phase extraction column, activating and balancing the micro solid phase extraction column with an activation solution and a balancing solution, dissolving the protein hydrolysate in the balancing solution, and transferring the glycopeptide enrichment material to the protein hydrolysate in a mass ratio of 100:1 to 1000:1; 2) washing the micro solid phase extraction column with an organic solution having a pH value of 1-7 and a volume of 5-200 times the column volume; 3) Using 5-100 column volumes and an eluent with a pH value of 1-12 for elution to obtain glycopeptides.

3. The method for simultaneous enrichment of N-linked glycopeptides and O-linked glycopeptides according to claim 1, characterized in that: The steps of separating and enriching glycopeptides using the dispersed solid phase extraction mode include: 1) activating the glycopeptide enriched material with an activation solution, mixing the glycopeptide enriched material with a protease hydrolysate at a mass ratio of 100:1-1000:1, incubating for 0.5 minutes to 12 hours, separating by filtration or centrifugation, discarding the supernatant, and collecting the precipitate; 2) washing the precipitate with an organic solution having a pH value of 1-7; 3) The washed precipitate is eluted with an eluent having a pH value of 1-12, separated by filtration or centrifugation, and the supernatant is collected to obtain the glycopeptide.

4. The method for simultaneous enrichment of N-linked glycopeptides and O-linked glycopeptides according to claim 2 or 3, characterized in that: The organic solution, the activation solution and the balancing solution are all mixed solutions of organic solvent, organic acid and water, and the eluent contains an alkaline solvent.

5. The method for simultaneous enrichment of N-linked glycopeptides and O-linked glycopeptides according to claim 4, characterized in that: The organic solvent is acetonitrile, methanol or ethanol, the volume concentration of the organic solvent is 50%-95%, the organic acid is formic acid, acetic acid or trifluoroacetic acid, the volume concentration of the organic acid is 0.1-5%.

6. The method for simultaneous enrichment of N-linked glycopeptides and O-linked glycopeptides according to claim 4, characterized in that: The volume concentration of the organic solvent in the activation solution is 10%-50%, and the pH value is 1-7.

7. The method for simultaneous enrichment of N-linked glycopeptides and O-linked glycopeptides according to claim 4, characterized in that: The volume concentration of the organic solvent in the equilibrium solution is 50%-95%, and the pH value is 1-7.

8. The method for simultaneous enrichment of N-linked glycopeptides and O-linked glycopeptides according to claim 4, characterized in that: The volume concentration of the alkaline solvent in the eluent is 0.1%-30%, and the pH value is 8-12.

9. The method for simultaneous enrichment of N-linked glycopeptides and O-linked glycopeptides according to claim 4, characterized in that: The volume concentration of the alkaline solvent in the eluent is 0.1%-5%, and the pH value is 1-7.

10. A method for simultaneous identification of N-linked glycopeptides and O-linked glycopeptides, characterized in that: Used for identifying the glycopeptide-enriched material obtained by the simultaneous enrichment of N-linked glycopeptides and O-linked glycopeptides in the method according to any one of claims 1 to 9, the method comprising: Using glycoproteomics retrieval software, by adding detailed information of N-glycan and O-glycan sugar libraries in the sugar library at the same time, setting the enzyme cleavage method and modification type that match the experimental conditions, and then using simultaneous retrieval to simultaneously identify N-linked glycopeptides and O-linked glycopeptides.

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