One-Step Preparation of Zwitterionic-Rich Hydrophilic Hydrothermal

May 30, 2019 - The water used in all experiments was doubly distilled and purified with a Milli-Q system (Millipore Inc., Milford, MA, USA). The fused...
0 downloads 0 Views 13MB Size
Download PDF
Research Article Cite This: ACS Sustainable Chem. Eng. 2019, 7, 11511−11520

pubs.acs.org/journal/ascecg

One-Step Preparation of Zwitterionic-Rich Hydrophilic Hydrothermal Carbonaceous Materials for Enrichment of N‑Glycopeptides Xiaowei Li,†,§ Haiyang Zhang,†,‡ Na Zhang,†,§ Shujuan Ma,†,‡ Junjie Ou,*,† and Mingliang Ye†

Downloaded via GUILFORD COLG on July 19, 2019 at 05:35:29 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), 457 Zhongshan Road, Dalian 116023, China ‡ Key Laboratory of Synthetic and Natural Function Molecule Chemistry of Ministry of Education, College of Chemistry and Materials Science, Northwest University, Xi’an 710069, China § University of Chinese Academy of Sciences, Beijing 100049, China S Supporting Information *

ABSTRACT: Two kinds of novel zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) materials decorated with phosphorylcholine or sulfobetaine type zwitterionic groups were facilely synthesized employing hydrothermal carbonization (HTC) reaction, in which sustainable carbohydrate (glucose) could be converted into functionalized carbonaceous materials with strong hydrophilicity using a one-step and environmentally mild process. The properties of obtained materials were characterized by helium ion microscopy (HIM), N2 adsorption/desorption measurement, static water contact angle measurement, elemental analysis, Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), and ζpotential. As a result, 26 N-glycopeptides with S/N above 20 could be detected from 150 fmol IgG digests after hydrophilic interaction chromatography (HILIC) enrichment by using as-synthesized material HTC-Glc-10%MPC. The limit of detection in matrix assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF MS) analysis was as low as 5 fmol and enrichment selectivity reached 100:1. Furthermore, 175 N-glycosylation sites in 285 Nglycopeptides from 92 glycoproteins were identified from 2 μL of human serum. This work provided a new, simple, and green way to synthesize an N-glycopeptide enrichment material for liquid chromatography−mass spectrometry/mass spectrometry (LC-MS/MS) analysis of glycoproteomes. KEYWORDS: Glucose-derived, Hydrothermal, Zwitterionic hydrophilic interaction liquid chromatography, ZIC-HILIC, Liquid chromatography−mass spectrometry/mass spectrometry, LC-MS/MS, N-Glycopeptide, Enrichment

■

INTRODUCTION As one of the most important post-translational modifications, protein glycosylation occurs on more than half of human proteins according to the records and predictions of the SwissProt database.1 Glycoproteins have been proven to participate in many important cellular activities and disease processes, such as cell communication, signaling, and cell adhesion. Besides, many glycoproteins have been recognized as clinical biomarkers and therapeutic targets in studies.2,3 Given all of the above, the study of protein glycosylation is of great importance in the field of life sciences. Glycoproteomics research mainly focuses on glycosylation sites, glycan structure, glycan composition, and the effect of glycosylation on protein function. However, the analysis of glycopeptides is a great challenge owing to three facts. First, glycopeptides account for only 2−5% of peptides because of the existence of high abundance proteins.4 Second, the existence of glycans can drastically reduce the ionization efficiency of glycopeptides. Third, different glycoforms occupying a given glycosylation site © 2019 American Chemical Society

give rise to a microheterogeneity, which dramatically decreases the abundance of each individual species. All of these reasons lead to extremely low signals compared with those of nonglycosylated peptides and make N-glycopeptide analysis a challenging task. Therefore, the enrichment of glycopeptides before MS analysis is of great important for glycoproteomics research. The conventional enrichment mechanisms in glycoproteomics mainly include four types: hydrophilic interaction liquid chromatography (HILIC),5,6 lectin affinity chromatography,7 boronic acid-assisted enrichment,8 and hydrazide chemistry.9 Among them, HILIC has attracted more and more attention from researchers due to many advantages, such as high throughput, low biases toward different glycopeptides, and good reproducibility.10 Consequently, a number of HILIC Received: March 8, 2019 Revised: May 25, 2019 Published: May 30, 2019 11511

DOI: 10.1021/acssuschemeng.9b01382 ACS Sustainable Chem. Eng. 2019, 7, 11511−11520

Research Article

ACS Sustainable Chemistry & Engineering

Figure 1. (a) HILIC enrichment process of glycopeptides and one-step synthesis process diagrams of (b) HTC-Glc-x%MPC and (c) HTC-Glc-x% MSA.

source, and small amounts of organic monomers are required in order to provide functional groups.25 Since hydrothermal carbonization operates with biomass, it is regarded as a sustainable “CO2-neutral” or even “CO2-negative” operation if microbial degradation of biomass is taken into consideration.26,27 Although the hydrothermal carbonization process of glucose to produce carbon spheres was established a long time ago and has been widely applied in adsorption,25 catalysts,28 and electrode materials,29 to the best of our knowledge, its application as stationary phase in liquid chromatography was seldom explored. Herein, we proposed the production of carbon microspheres rich in phosphorylcholine or sulfobetaine type zwitterionic groups using one step hydrothermal carbonization of glucose in the presence of 2-methacryloyloxyethyl phosphorylcholine (MPC) or [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl) ammonium hydroxide (MSA). The resulting materials were applied for enrichment of Nglycopeptides in ZIC-HILIC mode owing to their highly hydrophilic surface.

materials were synthesized to capture hydrophilic glycopeptides through hydrophilic−hydrophilic interactions by introducing more hydrophilic groups such as carboxyl groups,11 amino groups, 12 hydroxyl groups, 13 and zwitterionic groups14−16 onto the surface of materials. Several carriers such as mesoporous silica,11,17,18 magnetic nanoparticles,19,20 metal−organic frameworks,21,22 covalent organic frameworks,23 and macroporous adsorption resins24 have been used as substrates for grafting functional groups. Nevertheless, the preparation process was generally complicated and timeconsuming and involved toxic organic reagents. For example, the synthesis process of KS-TC-TBHP-SPE, a most popular ZIC-HILIC material, was troublesome and took more than 48 h.16 In this regard, a new HILIC material with facile functionalization route is desired. Compared with conventional modification of carbon materials with harsh conditions above 800 °C and organic compounds, the hydrothermal carbonization process is a mild, general, and facile process in which functional carbonaceous materials can be produced under 180 °C via only one step. Usually, a cheap and widely available carbohydrate (e.g., glucose, sucrose, or cellulose) was chosen as the main carbon 11512

DOI: 10.1021/acssuschemeng.9b01382 ACS Sustainable Chem. Eng. 2019, 7, 11511−11520

Research Article

ACS Sustainable Chemistry & Engineering

■

serum, 10 mg of HTC-Glc-MPC were incubated with tryptic digests of 2 μL of human serum, and the enriched N-glycopeptides from human serum were deglycosylated. Other experimental procedures were same as the above protocols. Deglycosylation of N-Glycopeptides by PNGase F. After enrichment, the N-glycopeptides were lyophilized and redissolved in 60 μL of 10 mM NH4HCO3 solution with 0.5 μL of PNGase F (pH = 8.0) at 37 °C for 18 h to remove the glycan moieties. Then the deglycosylated peptides were analyzed by MALDI-TOF MS or quenched by pure FA and lyophilized before LC-MS/MS analysis. Recovery Test of N-Glycopeptide Enrichment. The recovery of material toward N-glycopeptides was assessed according to a previously reported strategy.30,31 In brief, an equal amount of IgG tryptic digests was labeled with light and heavy isotopes. Then, the heavy-tagged IgG tryptic digests were enriched with 5 mg of materials. The enriched heavy-tagged N-glycopeptides were mixed with the light-tagged IgG tryptic digests. After that, the mixture was reenriched with 5 mg of materials. Finally, the eluted N-glycopeptides were deglycosylated for MALDI-TOF MS analysis. The recoveries were calculated by the peak intensity ratios of the heavy-tagged glycopeptides to the light-tagged glycopeptides. Mass Spectrometry Analysis and Database Searching. MALDI-TOF MS experiments were carried out with a 5800 Proteomics Analyzer (Applied Biosystems, USA) with a pulsed Nd/ YAG laser at 355 nm in reflective positive ion mode. Obtained glycopeptide solution (0.5 μL ) and 0.5 μL of DHB solution (25 mg/ mL DHB in ACN/H2O/H3PO4, 70/29/1, v/v/v) were spotted on the MALDI plate for MS analysis. LC-MS/MS analysis was carried out using a Dionex UltiMate 3000 Nano LC system coupled with a Q-Exactive mass spectrometer (Thermo Fisher Scientific, USA). The deglycosylated peptides mixture was redissolved in FA/H2O (0.1/99.9, v/v) and loaded on a trap column (2 cm × 150 μm i.d.) packed with C18 AQ beads (1.9 μm, 120 Å, Dr. Maisch Gmbh, Germany). Then the peptides were separated on a capillary analysis column (12 cm × 75 μm i.d.) packed with the same C18 AQ beads as the analytical column. Mobile phases A (H2O/FA, 99.9/0.1, v/v) and B (ACN/FA, 99.9/0.1, v/v) were used for RPLC separation. The gradient elution was programmed as follows: 0% B for 18 min, 9−35% B in 90 min, 35−45% B in 13 min, 45−90% B in 2 min, 90% B for 10 min, 4% B in 15 min. All LC-MS/MS raw data were searched with Mascot Daemon v2.3.0 against a database. The data were treated by ArMone v2.0. The cutoff false discovery rate (FDR) was controlled below 1%, and ion score was greater than 20 for identified peptides. The mass tolerances were 10 ppm for initial precursor ions and 0.05 Da for fragment ions. Two missed cleavages were permitted for trypsin restriction. Cysteine carbamidomethylation (+57.0215 Da) was set as fixed modification; asparagine and glutamine deamidation (+0.9840 Da) and methionine oxidation (+15.9949 Da) were set as variable modifications. Only peptides with N-!P-S/T were considered as N-linked glycopeptides. The N-glycosylation site was also required to contain a modification of asparagine deamidation. All LC-MS/MS data have been deposited into the jPOST database (http://jpostdb.org/), accession code JPST000593/PXD013709. Characterization. The morphology study of carbon microspheres was carried out on HIM (ORION NANOFAB, Germany). For specific surface area analysis, the resulting materials were degassed at 120 °C over 6 h. Then, N2 adsorption/desorption experiments were carried out at 77 K using ASAP 2460 Physisorption Analyzer (Micromeritics, USA). The BET surface area was calculated using the Brunauer−Emmett−Teller (BET) equation. Water contact angles were measured on a DSA 100 machine (KRUSS, Hamburg, Germany) with 5 μL of water drop after the obtained powders were prepared into tablets under 8 MPa. Elemental analysis of nitrogen and sulfur were carried out on FLASH EA 1112 Elemental Analyzer (Thermo, USA). Elemental analysis of phosphorus was carried out on ICP-OES 7300DV (PerkinElmer, USA). FT-IR spectra were obtained on a TENSOR 27 spectrometer with KBr pellets containing 1 wt % of the resulting sample (Bruker Optics, Germany). XPS was performed on ESCALAB 250 (Thermofisher, USA) with a

EXPERIMENTAL SECTION

Chemicals and Materials. MPC (95%), MSA (95%), trifluoroacetic acid (TFA, 99%), formic acid (FA, 98%), 2,5-dihydroxybenzoic acid (DHB, 98%), dithiothreitol (DTT, 99%), iodoacetamide (IAA, 99%), immunoglobulin G (IgG, 95%), bovine serum albumin (BSA, 98%), trypsin, ammonium hydrogen carbonate (NH4HCO3), and urea were obtained from Sigma-Aldrich (St. Louis, MO, USA). Glucose (99.5%) was obtained from Aladdin (Shanghai, China). Acetonitrile (ACN) and methanol (CH3OH) were HPLC-grade and obtained from Merck (Darmstadt, Germany). Phosphoric acid (H3PO4), hydrochloric acid (HCl), and sodium hydroxide (NaOH) were obtained from Tianjin Kermel Chemical Plant (Tianjin, China). The water used in all experiments was doubly distilled and purified with a Milli-Q system (Millipore Inc., Milford, MA, USA). The fused silica capillaries with inner diameter (i.d.) of 75 and 150 μm were purchased from Polymicro Technologies (Phoenix, AZ). Preparation of Hydrophilic Carbonaceous Materials. A determined amount of MPC was dissolved into glucose aqueous solution at a concentration of 10% (w/v) until the concentration of MPC was 3%, 5%, and 10% (w/v) in the initial solution. The solution was then transferred into a Teflon inlet autoclave encapsulated within a stained steel container and heated in an oven at 180 °C for 6 h. Then the resulting materials were washed thoroughly with water, ethanol, and ACN to remove all byproducts and unreacted molecules until the supernatant was colorless, and the product was dried in a vacuum drying oven at 60 °C overnight. The obtained materials were denoted as HTC-Glc-x%MPC, where x represents the percentage of MPC in the initial solution. For example, the material synthesized from a solution with a concentration of MPC was 10% (w/v) was named as HTC-Glc-10%MPC. Similarly, the detailed procedures for synthesis of MSA-modified carbonaceous materials were the same as those for HTC-Glc-x%MPC. The resulting materials were denoted as HTC-Glc-x%MSA, where x represents the percentage of MSA in the initial solution. Different amounts of MSA were added into 10% (w/v) glucose solution until the concentration (w/v) of MSA was 3%, 5%, and 10% in the original solution. For example, the resulting material from 3% (w/v) MSA was called as HTC-Glc-3%MSA. In addition, a control material named HTC-Glc was also prepared from 10% (w/v) glucose solution without adding any MPC or MSA. Tryptic Digestion of Proteins. Tryptic digestion process of proteins including human IgG, BSA, and human serum was carried out according to the reported method with minor modification.17 Two milligrams of proteins were dissolved in 0.5 mL denaturing solution (100 mM NH4HCO3, 8 M urea, pH = 8.2). After centrifugation, samples were reduced with 10 μL of 1 M DTT at 37 °C for 2 h and alkylated by 20 μL of 1 M IAA at room temperature under exclusion of light for 40 min. Then, 3.5 mL of NH4HCO3 solution (100 mM) were added in order to dilute urea in denaturing solution to 1 M. Finally, the solution was incubated with 80 mg of trypsin in 37 °C water bath for 18 h. Tryptic digests were then desalted, divided into several parts, lyophilized, and stored at −20 °C for further use. Enrichment of N-Glycopeptides with HILIC. HILIC enrichment process (Figure 1a) was carried out according to following protocol: 5 mg of materials were first dispersed and equilibrated in 400 μL of loading solution (ACN/H2O/TFA, 80/19/1, 85/14/1, or 90/9/1, v/v/v) for 20 min. After removal of the supernatant by centrifugation, 9.0 μg of tryptic digests of human IgG were redissolved in 400 μL of loading solution (consistent with the solution used in the above equilibrium process), and then the solution was added and incubated under 1500 rpm at 25 °C for 1 h. After that, the ZICHILIC material was separated from the solution by centrifugation and washed with 400 μL of loading solution three times (10 min each time) to remove nonspecifically adsorbed nonglycopeptides. Finally, the enriched N-glycopeptides were released with 200 μL of elution solution (ACN/H2O/TFA, 30/69/1, v/v/v) under 1500 rpm for 40 min and then analyzed directly by MALDI-TOF MS or lyophilized and deglycosylated. For enriching N-glycopeptides from human 11513

DOI: 10.1021/acssuschemeng.9b01382 ACS Sustainable Chem. Eng. 2019, 7, 11511−11520

Research Article

ACS Sustainable Chemistry & Engineering

Figure 2. HIM images of (a) HTC-Glc, (b) HTC-Glc-3%MPC, (c) HTC-Glc-5%MPC, (d) HTC-Glc-10%MPC, (e) HTC-Glc-3%MSA, (f) HTCGlc-5%MSA, and (g) HTC-Glc-10%MSA.

Figure 3. Water contact angles of (a) HTC-Glc-3%MPC, (b) HTC-Glc-5%MPC, (c) HTC-Glc-10%MPC, (d) HTC-Glc-3%MSA, (e) HTC-Glc5%MSA, and (f) HTC-Glc-10%MSA. monochromatized Al Kα X-ray source. The ζ-potentials of carbon microspheres in water (0.1 g/L; 0.1 M NaOH and 0.1 M HCl were used to adjust pH) were measured at 25 °C with a Zetasizer Nano ZS90 (Malvern Instruments Ltd., Malvern, U.K).

derivatives like 5-(hydroxymethyl)-2-furaldehyde and furfural aldehyde as intermediates. Then the intermediates further underwent a series of cycloadditions and condensation reactions.35,36 The final carbonaceous scaffold contains the furan unit as the main repeating motif.37 Lastly, furans and their derivatives were involved in Diels−Alder type cycloaddition in the presence of dienophiles such as MSA or MPC. Linear polymerization of dienophiles also can be excluded.25,38 As a result, two kinds of hydrophilic carbonaceous microspheres would be formed via one-step hydrothermal reaction. On the basis of HIM images, HTC-Glc synthesized without adding any zwitterionic monomers was monodispersed microspheres with a size of around 200 nm (Figure 2a). After zwitterionic MPC was added into glucose solution, HTC-Glc3%MPC had a bigger particle size of around 1 μm (Figure 2b). The particle size of HTC-Glc-x%MPC became smaller with an increase of MPC concentration, as those of HTC-Glc-5%MPC (Figure 2c) and HTC-Glc-10%MPC (Figure 2d) were 400 and 200 nm, respectively. These results might be due to the fact that more MPC molecules increased the chance of contact with furans and their derivatives, leading to the premature occurrence of cycloaddition.25,38 As shown in Figure 2e, HTC-

■

RESULTS AND DISCUSSION Preparation and Characterization of HTC-Glc-x% MPC, HTC-Glc-x%MSA, and HTC-Glc. The synthetic procedures for HTC-Glc-x%MPC and HTC-Glc-x%MSA are presented in Figure 1b,c. MPC is a zwitterionic monomer containing both a positively charged quaternary ammonium group and a negatively charged phosphoric group. Similarly, MSA is another zwitterionic monomer, which contains quaternary ammonium groups and sulfonic groups. In order to obtain carbonaceous materials with high hydrophilicity, biomass glucose was selected as the carbon source, and MPC or MSA was chosen as dienophile and functional reagent. Then, a hydrothermal reaction under 180 °C was conducted to decorate zwitterionic functional groups onto the surface of carbon spheres. A possible reaction mechanism has been proposed according to the previous reports.32−34 In detail, glucose was first dehydrated to produce furfural and furfural 11514

DOI: 10.1021/acssuschemeng.9b01382 ACS Sustainable Chem. Eng. 2019, 7, 11511−11520

Research Article

ACS Sustainable Chemistry & Engineering

bands at 1700 and 1620 cm−1 in HTC-Glc (Figure 4b), HTCGlc-10%MPC (Figure 4a), and HTC-Glc-3%MSA (Figure 4d), which were attributed to CO and CC stretching vibrations, respectively. These results support the concept of aromatization of glucose during hydrothermal treatment.39 Comparing the FT-IR spectra of MPC (Figure 4c), HTC-Glc (Figure 4b), and HTC-Glc-10%MPC (Figure 4a), we also could see the peaks at around 1240 and 1080 cm−1 in MPC and HTC-Glc-10%MPC corresponding to the stretching vibration of PO and the stretching vibration of −POCH2−, as well as the peaks at around 1170 and 970 cm−1 corresponding to the stretching vibration of C−N and N+(CH3)3.40 As for HTC-Glc-3%MSA, the sulfobetaine structure was confirmed by the presence of −SO3 at around 1043 cm−1 and −N+(CH3)2− at around 960 cm−1 in MSA (Figure 4e) and HTC-Glc-3%MSA (Figure 4d) according to the previously reported literature.41,42 At the same time, the above peaks did not appear in HTC-Glc (Figure 4b), also confirming the successful grafting of two kinds of zwitterionic groups. To investigate the elemental composition and chemical states of some elements on the surface of HTC-Glc-10%MPC and HTC-Glc-3%MSA, C 1s, O 1s, N 1s, P 2p, and S 2p scanning spectra were obtained by XPS (Figure 5). The full spectrum of HTC-Glc-10%MPC (Figure 5a) and HTC-Glc3%MSA (Figure 5b) confirmed the existence of N and P atoms in HTC-Glc-10%MPC, as well as the existence of N and S atoms in HTC-Glc-3%MSA. In the high-resolution N 1s spectrum (Figure 5c,e), quaternary N could be identified at 402.05 and 402.7 eV.43 The P 2p spectrum of HTC-Glc-10% MPC is shown in Figure 5d. The peak at 134.01 eV was assigned to P−O.44 The S 2p spectrum of HTC-Glc-3%MSA in Figure 5f could be attributed to −SO3 corresponding to the peak at 167.85 eV.43,45,46 It can be concluded from XPS analysis that the surface of the two carbonaceous materials was covered with zwitterionic groups. The surface charge properties were studied, and the results are shown in Figure 6. In detail, ζ-potentials of HTC-Glc-10% MPC, HTC-Glc-3%MSA, and HTC-Glc were measured at four pH levels (pH = 3, 5, 9, and 11). It was found that the three kinds of materials had an overall negative surface charge above pH 3, which might be ascribed to the hydroxyl and carboxyl groups located at the surface25 and in good agreement with the results in previous publications.16,40 In addition, the absolute values of ζ-potential of HTC-Glc-10%MPC and HTC-Glc-3%MSA were lower than that of HTC-Glc at the same pH and increased with an increase of pH. These results demonstrated that adding two kinds of zwitterionic groups to the initial mixture could decrease the charge density of the surface of HTC-Glc, also indicating the successful grafting of zwitterionic functional groups. Specific Enrichment of N-Glycopeptides and Optimization of Enrichment Performance by HTC-Glc-10% MPC, HTC-Glc-3%MSA, and HTC-Glc. Taking advantage of good hydrophilicity of the obtained carbonaceous materials, we applied HTC-Glc-10%MPC and HTC-Glc-3%MSA for selective enrichment of N-glycopeptides from tryptic digests of human IgG in HILIC mode. The hydrophilic partitioning occurs between a water-enriched layer on the surface of a hydrophilic stationary phase and a relatively hydrophobic eluent solution.47 Therefore, the concentration of ACN in the mobile phase plays a significant role in the glycopeptide enrichment with HILIC materials. In this work, three kinds of

Glc-3%MSA was monodispersed microspheres with size of around 500 nm, while HTC-Glc-5%MSA and HTC-Glc-10% MSA were amorphous owing to serious aggregation of the particles. The porosities of the six carbonaceous materials were determined by N2 adsorption−desorption measurement. Based on the BET model, the surface areas were 7.36, 5.28, 8.55, 6.75, 3.85, and 2.47 m2/g for HTC-Glc-3%MPC, HTC-Glc-5% MPC, HTC-Glc-10%MPC, HTC-Glc-3%MSA, HTC-Glc-5% MSA, and HTC-Glc-10%MSA, which was as low as other hydrothermal carbons.25 To evaluate the hydrophilicity of the two kinds of materials, static water contact angles of HTC-Glc-x%MPC and HTCGlc-x%MSA were measured. As shown in Figure 3, water contact angles of HTC-Glc-3%MPC (Figure 3a), HTC-Glc-5% MPC (Figure 3b), HTC-Glc-10%MPC (Figure 3c), HTC-Glc3%MSA (Figure 3d), HTC-Glc-5%MSA (Figure 3e), and HTC-Glc-10%MSA (Figure 3f) were 34.5°, 32.9°, 26.6°, 27.6°, 37.7°, and 38.1°, respectively, which indicated that all the obtained carbonaceous materials possessed strongly hydrophilic surfaces. The contact angle of HTC-Glc-x%MPC tended to decrease as the content of MPC in the initial solution increased. However, the serious aggregation phenomenon may cause bigger contact angles of HTC-Glc-5%MSA and HTCGlc-10%MSA than HTC-Glc-3%MSA, although the former two should have the stronger hydrophilicity owing to higher content of MSA in the initial solution. Considering the morphology and hydrophilicity of these carbonaceous materials, HTC-Glc-10%MPC and HTC-Glc-3%MSA were selected for the following N-glycopeptide enrichment, and the remaining characterizations were also mostly concerning HTCGlc-10%MPC and HTC-Glc-3%MSA. Elemental analysis results of HTC-Glc, HTC-Glc-10%MPC, and HTC-Glc-3%MSA are shown in Table 1. Prior to the Table 1. Results of Elemental Analysis of HTC-Glc, HTCGlc-10%MPC, and HTC-Glc-3%MSAa HTC-Glc HTC-Glc-10%MPC HTC-Glc-3%MSA

NEA (%)

PICP‑OES (%)

SEA (%)

N/P

N/S