Covalent organic framework functional materials and their applications in glycopeptide enrichment

doi:10.3724/SP.J.1123.2021.02001

Abstract

Abstract:

Protein glycosylation is among the most important post-translational modifications in living organisms and the research in the field of protein glycosylation continues to garner attention. Currently, the efficient separation and enrichment of glycoproteins and glycopeptides is the primary challenge of glycoproteomics research. The number of glycoproteins is small in complex biological samples. Moreover, the presence of highly-abundant, non-glycosylated, and modified peptides makes the detection of low-abundance glycopeptides more difficult. Therefore, efficient glycopeptide enrichment methods are required to improve the detection of these compounds. The development of highly selective glycopeptide enrichment tools is important to efficiently capture glycoproteins or glycopeptides at the molecular level. Compared with traditional glycopeptide-enriched materials, covalent organic framework materials have the advantages of large specific surface area and rich modification sites, thereby exhibiting great application potential in the field of glycopeptide enrichment. In this study, a novel covalent organic framework material (O-T-D-COFs) was prepared and applied for selective glycopeptide enrichment. We applied the solvothermal method, using 2,5-dimethoxy benzene-1,4-2 formaldehyde and 1,3,5-Tris(4-aminophenyl) benzene, to synthesize imino-based COFs. The Schiff base generated via copolymerization condensation reaction constitutes the framework of the material. Next, the synthesized intermediate material was oxidized to improve the enrichment performance of the material. The functional, specific glycopeptide-binding groups were modified on the COF channels and the structure of the material was characterized using scanning and transmission electron microscope, as well as infrared spectrum and solid-state nuclear magnetic resonance. The enrichment conditions comprised the loading and elution steps, including the optimization of the elution conditions. We could observe the clear profile of 32 glycopeptides derived from human serum immunoglobulin G (IgG) tryptic digests with a significantly improved signal-to-noise (S/N) ratio. We applied a complex sample system to verify the enrichment selectivity of the material when the molar ratios of the IgG and bovine serum albumin (BSA) tryptic digest mixtures reached 1∶50. In addition, we investigated the enrichment performance of the detection limit, enrichment capacity, recovery rate of the material, and the application potential in glycopeptides enrichment using real samples. The material showed a good detection limit (2.5 fmol/μL), an ideal enrichment capacity (120 mg/g), and enrichment recovery (103.5%±6.6% and 101.5%±10.4%). We identified a total of 86 glycopeptides derived from 53 glycoproteins with 94 N-glycosylation sites from only 1 μL human serum. The O-T-D-COFs exhibited a good glycopeptide separation and enrichment potential, indicating that the COF material has promising application potential in glycoproteomics.

Key words: mass spectrometry (MS), covalent organic framework (COF) material, functional modification, glycopeptides, selective enrichment

CLC Number:

O658

SHENG Qianying, ZHOU Yang, ZHAO Zhiquan, WANG Yaohui, LI Weicheng, KE Yanxiong, LAN Minbo, QING Guangyan, LIANG Xinmiao. Covalent organic framework functional materials and their applications in glycopeptide enrichment[J]. Chinese Journal of Chromatography, 2021, 39(6): 588-598.

Figures/Tables 10

Fig. 1 Synthetic route of O-T-D-COF

Fig. 2 SEM images of O-T-D-COF at (a) 5000 and (b) 18000 magnifications, and (c)13C NMR spectra and (d) IR spectra of O-T-D-COF

Fig. 3 TEM images of O-T-D-COF

Fig. 4 Effects of (a) loading buffer acidity, (b) ACN volume percentage in loading buffer, (c) washing buffer acidity, and (d) eluent buffer acidity on enrichment m/z 2601.2, 2633.2, 2764.1, 2795.5, 2925.6, and 2957.5: six highly abundant glycopeptides of IgG digests.

Fig. 5 Mass spectra of the tryptic digest of IgG (a) before and (b) after enrichment under optimum conditions m/z of glycopeptide peak No. 1: 2398.5; 2: 2430.4; 3: 2455.5; 4: 2487.5; 5: 2560.5; 6: 2592.5; 7: 2601.2; 8: 2617.5; 9: 2633.2; 10: 2642.4; 11: 2649.5; 12: 2659.0; 13: 2691.0; 14: 2764.1; 15: 2779.6; 16: 2795.5; 17: 2804.5; 18: 2811.5; 19: 2821.3; 20: 2836.5; 21: 2853.3; 22: 2925.6; 23: 2957.5; 24: 2966.6; 25: 2983.2; 26: 2998.5; 27: 3015.1; 28: 3054.5; 29: 3129.2; 30: 3160.5; 31: 3216.6; 32: 3248.6.

Fig. 6 Mass spectra of the complex system for glycopeptides enrichment Mixture of tryptic digests of human serum immunoglobulin G (IgG) and bovine serum albumin (BSA) at amount of material ratios of (a) 1∶10 and (b) 1∶50. Peak Nos.: see Fig. 5.

Fig. 7 Mass spectra of the tryptic digests of (a) 5 fmol/μL and (b) 2.5 fmol/μL IgG Peak Nos.: see Fig. 5.

Fig. 8 Results of enrichment of 6 μg IgG tryptic digest with different amount of O-T-D-COF materials m/z 2601.2, 2633.2, 2764.1, 2795.5, 2925.6, and 2957.5: glycopeptides.

Fig. 9 Three paralleled experiments of the recovery of the light- and heavy-dimethyl labeled deglycosylated peptides from human IgG tryptic digest

Table 1 Recoveries of two deglycosylated peptides from human IgG tryptic digest enriched by O-T-D-COF (n=3)

No.	Recoveries/%
No.	EEQFN#STFR (m/z 1158.5)	EEQYN#STYR (m/z 1190.5)
1	110.1	93.8
2	99.0	111.9
3	101.4	98.8
Average	103.5±6.6	101.5±10.4

References

[1]	Feng X, Ding X S, Jiang D L. Chem Soc Rev, 2012,41(18):6010
[2]	Geng K Y, He T, Liu R, et al. Chem Rev, 2020,120(16):8814
[3]	Ding S Y, Wang W. Chem Soc Rev, 2013,42(2):548
[4]	Huang N, Wang P, Jiang D. Nat Rev Mater, 2016,1(10):16068
[5]	Liang R R, Jiang S Y, Zhao X. Chem Soc Rev, 2020,49(12):3920
[6]	Wang X, Ye N S. Electrophoresis, 2017,38:3059
[7]	Chen X, Geng K, Liu R, et al. Angew Chem Int Edit, 2020,59(13):5050
[8]	Mann M, Jensen O N. Nat Biotechnol, 2003,21(3):255
[9]	Huang J F, Wang F J, Ye M L, et al. J Chromatogr A, 2014,1372:1
[10]	Zhang Y, Zhang C, Jiang H C, et al. Chem Soc Rev, 2015,44(22):8260
[11]	Dube D H, Bertozzi C R. Nat Rev Drug Discov, 2005,4(6):477
[12]	Hart G W, Copeland R J. Cell, 2010,143:672
[13]	Moremen K W, Tiemeyer M, Nairn A V. Nat Rev Mol Cell Bio, 2012,13(7):448
[14]	Ongay S, Boichenko A, Govorukhina N, et al. J Sep Sci, 2012,35(18):2341
[15]	Gaunitz S, Nagy G, Pohl N L B, et al. Anal Chem, 2017,89(1):389
[16]	Qing G Y, Yan J Y, He X N, et al. Trend Anal Chem, 2020,124:115570
[17]	Pan S, Chen R, Aebersold R, et al. Mol Cell Proteomics, 2011, 10(1): R110.003251
[18]	Liu S, Jiang X T, Shang Z, et al. Anal Chim Acta, 2020,1123:18
[19]	Witze E S, Old W M, Resing K A. Nat Meth, 2007,4(10):798
[20]	Cummings R D, Pierce J M. Chem Biol, 2014,21(1):1
[21]	Palaniappan K K, Bertozzi C R. Chem Rev, 2016,116(23):14277
[22]	Huang B Y, Yang C K, Liu C P, et al. Electrophoresis, 2014,35(15):2091
[23]	Qing G Y, Lu Q, Xiong Y T, et al. Adv Mater, 2017,29(20):1604670
[24]	Cai T P, Rahman A F M M, et al. Microchim Acta, 2017,184(8):2629
[25]	Chen Y X, Sheng Q Y, Hong Y Y, et al. Anal Chem, 2019,91(6):4047
[26]	Chen C, Kang H J, Zhang X F, et al. Chinese Journal of Chromatography, 2019,37(8):845
[26]	陈成, 康虹健, 张小菲, 等. 色谱, 2019,37(8):845
[27]	Zheng H J, Jia J X, Li Z, et al. Anal Chem, 2020,92(3):2680
[28]	Lu Q, Chen C, Xiong Y T, et al. Anal Chem, 2020,92(9):6269
[29]	Zhang H Q, Lv Y Y, Du J, et al. Anal Chim Acta, 2020,1098:181
[30]	Zheng X T, Wang X, Zhang F S, et al. Chinese Journal of Chromatography, 2021,39(1):15
[30]	郑鑫彤, 王雪, 张福生, 等. 色谱, 2021,39(1):15
[31]	Ma Y F, Yuan F, Zhang X H, et al. Analyst, 2017,142(17):3212
[32]	Wang H P, Jiao F L, Gao F Y. J Mater Chem B, 2017,5(22):4052
[33]	Gao C H, Lin G, Lei Z X. J Mater Chem B, 2017,5(36):7496
[34]	Ding F J, Chu Z Y, Zhang Q Q, et al. Anal Chim Acta, 2019,1057:145
[35]	Wang J X, Li J, Gao M X, et al. Nanoscale, 2017,9(30):10750
[36]	Ma Y F, Wang L J, Zhou Y L, et al. Nanoscale, 2019,11(12):5526
[37]	Luo B, He J, Li Z Y, et al. ACS Appl Mater Interfaces, 2019,11(50):47218