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. 2017 Jan 13:14:3.
doi: 10.1186/s12014-017-9137-1. eCollection 2017.

Simultaneous analyses of N-linked and O-linked glycans of ovarian cancer cells using solid-phase chemoenzymatic method

Shuang Yang  1 , Naseruddin Höti  1 , Weiming Yang  1 , Yang Liu  1 , Lijun Chen  1 , Shuwei Li  2 , Hui Zhang  1
Affiliations

Simultaneous analyses of N-linked and O-linked glycans of ovarian cancer cells using solid-phase chemoenzymatic method

Shuang Yang et al. Clin Proteomics. .

Abstract

Background: Glycans play critical roles in a number of biological activities. Two common types of glycans, N-linked and O-linked, have been extensively analyzed in the last decades. N-glycans are typically released from glycoproteins by enzymes, while O-glycans are released from glycoproteins by chemical methods. It is important to identify and quantify both N- and O-linked glycans of glycoproteins to determine the changes of glycans.

Methods: The effort has been dedicated to study glycans from ovarian cancer cells treated with O-linked glycosylation inhibitor qualitatively and quantitatively. We used a solid-phase chemoenzymatic approach to systematically identify and quantify N-glycans and O-glycans in the ovarian cancer cells. It consists of three steps: (1) immobilization of proteins from cells and derivatization of glycans to protect sialic acids; (2) release of N-glycans by PNGase F and quantification of N-glycans by isobaric tags; (3) release and quantification of O-glycans by β-elimination in the presence of 1-phenyl-3-methyl-5-pyrazolone (PMP).

Results: We used ovarian cancer cell lines to study effect of O-linked glycosylation inhibitor on protein glycosylation. Results suggested that the inhibition of O-linked glycosylation reduced the levels of O-glycans. Interestingly, it appeared to increase N-glycan level in a lower dose of the O-linked glycosylation inhibitor. The sequential release and analyses of N-linked and O-linked glycans using chemoenzymatic approach are a platform for studying N-glycans and O-glycans in complex biological samples.

Conclusion: The solid-phase chemoenzymatic method was used to analyze both N-linked and O-linked glycans sequentially released from the ovarian cancer cells. The biological studies on O-linked glycosylation inhibition indicate the effects of O-glycosylation inhibition to glycan changes in both O-linked and N-linked glycan expression.

Keywords: Chemoenzymatic; Glycomics; Glycoprotein; Solid phase.

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Figures

Fig. 1
Fig. 1
Schematic diagram of sequential releases and analyses of N-linked and O-linked glycans via chemoenzymatic method. a Immobilize glycoproteins on solid support. b Modify sialic acids; c release N-glycans using PNGase F; d label N-glycans by the isobaric tags such as QUANTITY via reductive amination; e release O-glycans by β-elimination. The released O-glycans are purified using C18 cartridge and N-glycans are purified using Carbograph SPE column
Fig. 2
Fig. 2
Chemoenzymatic sequential releases of N-glycans and O-glycans from bovine serum-derived fetuin using GIG. a N-glycans were released by PNGase F on solid-phase; b O-glycans were released after N-glycans were released by mild β-elimination in 0.5 M PMP (1-phenyl-3-methyl-5-pyrazolone). The MS spectra was generated by MALDI
Fig. 3
Fig. 3
Sialylated O-glycans of mucin from bovine submaxillary glands (MBS) by MALDI-MS. a The sialic acids that were stabilized by carbodiimide coupling have a significantly increased MS signal; b the sialic acids without modification have low intensity in MALDI-MS. An internal standard (Neurotensin, 20 μM/1 μL) was spiked in the sample. The sialic acid modified glycans have one sodium adduct [Na]+, while native glycans have an extra sodium adduct per sialic acid
Fig. 4
Fig. 4
MS/MS fragmentation of QUANTITY-tagged N-glycans. The N4H5S2 was extracted from OVCAR-3 cells and labeled by QUANTITY. MS/MS was performed by Thermo Orbitrap Mass Spectrometer. When a reporter is lost, the mass is reduced by 176–178 with a “Loss reporter”
Fig. 5
Fig. 5
N-glycan profile of OVCAR-3 cells by LC–ESI–MS/MS. N-glycans were first released after sialic acid modification, and the released N-glycans were labeled using isobaric QUANTITY tags (Quaternary Amine Containing Isobaric Tag for Glycan). The labeled N-glycans were separated using a C18 analytical column (Thermo Scientific Acclaim PepMap, 15 cm). a Oligomannoses eluted from 0 to 10 min, b complex N-glycans eluted from 10 to 20 min, c Complex N-glycans eluted from 20 to 30 min, and d complex and sialylated N-glycans eluted from 30 to 40 min
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