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Letter
Recognition of bisecting N-Glycans on intact glycopeptides by two characteristic ions in low energy HCD spectra Liuyi Dang, Jiechen Shen, Ting Zhao, Fei Zhao, Li Jia, Bojing Zhu, Chen Ma, Dan-Qian Chen, Ying-Yong Zhao, and Shisheng Sun Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05639 • Publication Date (Web): 11 Apr 2019 Downloaded from http://pubs.acs.org on April 12, 2019
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Analytical Chemistry
Recognition of bisecting N-Glycans on intact glycopeptides by two characteristic ions in low energy HCD spectra
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Liuyi Dang†, Jiechen Shen†, Ting Zhao†, Fei Zhao‡, Li Jia†, Bojing Zhu†, Chen Ma†, Danqian Chen†, Yingyong Zhao†, Shisheng Sun†*
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†College
of Life Sciences, Northwest University, Xi’an 710069, China.
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‡College
of Fundamental Medicine, Shaanxi University of Chinese Medicine, Xianyang
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712046, China
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*Shisheng Sun, College of Life Science, Northwest University, Xi’an, Shaanxi province 710069, China. Email:
[email protected] 16
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ABSTRACT
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Bisecting N-glycan represents one of the most important modifications to the N-glycan core and
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it is involved in various biological processes. Despite many studies on the biological roles of
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bisecting N-glycans, current approaches for bisecting N-glycan analysis mainly rely on the use of
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the lectin PHA-E, which are of low specificity and sensitivity. Here, we described a
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straightforward method for the recognition of bisecting N-glycans on intact glycopeptides using
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two characteristic Y ions [peptide+HexNAc3Hex1] and [peptide+HexNAc3Hex1Fuc1] in low
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energy HCD MS/MS spectra. The critical aspect of the method is the combination use of low
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energy HCD fragmentation and intact glycopeptide analysis. With samples from rat renal tissues,
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we determined the optimal fragmentation energies and analyzed the influence of core fucosylation
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on the intensity of the [peptide+HexNAc3Hex1] ion. Using the method, we identified 183 intact
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glycopeptides with bisecting N-glycans and investigated the primary bisecting N-glycan structures
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and the possible biological roles of these identified proteins.
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Analytical Chemistry
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Bisecting N-glycan is characterized by a β1,4-linked GlcNAc residue attached to a β-mannose
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of the N-linked glycan core and the reaction is catalyzed by N-acetylglucosaminyltransferase-III
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(GnT-III/MGAT-III) 1. The bisecting N-glycans is known to play regulatory roles in biosynthesis
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of N-glycans, and its presence results in the suppression of further processing and elongation of N-
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glycans catalyzed by other glycosyltransferases (e.g., GnT-II, GnT-IV, GnT-V)2, 3. Previous
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reports have suggested that they are involved in a variety of biological functions such as cell-cell
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and cell-matrix interactions, cell growth control and tumor progression4-6.
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The expression of bisecting N-glycans is known to be tissue-specific (mostly expressed in
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kidney and brain). So far, different strategies have been used to identify and evaluate the
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expression of bisecting N-glycans in order to reveal their biological roles. Besides analyzing the
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expression of the enzyme GnT-III, the most used approach is detecting bisecting N-glycans by
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immune blotting using the lectin Phytohaemagglutinin-E (PHA-E)2, 6, 7. With the development of
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mass spectrometry, PHA-E has also been used for the enrichment of glycopeptides with bisecting
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N-glycans followed by mass spectrometry analysis, sometimes even with multilevel fragmentation
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for the determination of bisected GlcNAc 8, 9. However, the interaction of PHA-E with bisecting
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N-glycans actually requires not only the bisecting GlcNAc but also two galactose residues and the
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Manα1-6 arm. Moreover, PHA-E was also reported to bind weakly with non-bisected and
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galactosylated N-glycans at low temperature 10. All these drawbacks are detrimental to both the
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sensitivity and specificity of the methods using PHA-E for the analysis of bisecting N-glycans. In
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addition, a MS-based approach using an ion called D-221 (Gal-GlcNAc-Man-Man-loss of H2O)
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fragmentated from the bisecting N-glycans has also been reported for the identification bisecting
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N-glycans, which unfortunately only suitable for certain types of bisecting N-glycan 11. Therefore,
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simple and reliable methods are still needed for the investigation of bisecting N-glycans. 3
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Here, we report a novel method for the recognition of bisecting N-glycans on intact
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glycopeptides (Figure 1A). Normally, the identification of bisecting N-glycans can be achieved
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by recognizing the characteristic Y ion [peptide+HexNAc3Hex1, pep+N3H]. The characteristic Y
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ion [pep+N3H] can be generated due to cleavages between sugar chains by using a low HCD
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energy for the MS/MS fragmentation, which has been used to produce more diagnostic Y ions 12.
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The determination of the mass for [pep+N3H] ion is feasible thanks to the recently developed
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intact glycopeptide analysis 13, in which the sequence and mass of peptides as well as the glycan
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compositions can be obtained using software like GPQuest 14. Figure 1B shows a representative
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MS/MS spectrum of a peptide LHNQLLP510N#TTTVEK with bisecting N-glycosylation from
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glutathione hydrolase 1(GGT1)，one of the initial proteins that was known to be modified with
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bisecting N-glycan 15. As indicated, the peak with m/z=1190.586 (Y4 ion with 2+ charge, marked
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in red) represents the [pep+N3H] ion of the intact glycopeptide, which are specifically produced
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by the bisecting glycan. By detecting this characteristic ion, the bisecting N-glycans on intact
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glycopeptides can be identified.
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To determine the optimal energy for the bisecting N-glycan identification, we analyzed the
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energy-dependent breakdown of glycopeptides. A total of 34 spectra were first used for the
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analysis. Each of these spectra contained at least 3 out of 4 potential Y ions from bisecting N-
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glycans (pep+HexNAc1, pep+HexNAc2, pep+HexNAc2Hex1 and obligatory pep+N3H) but no
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pep+HexNAc3 ion (which could potentially come from a glycopeptide co-modified by both N- and
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O-linked glycans) to ensure the high confidence of bisecting glycopeptide identification. These
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spectra represented 19 bisecting glycopeptides that comprise of eight high abundant glycan
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structures (N5H3, N5H3F1, N5H4, N5H4S1, N5H5, N5H5F1, N5H6S1 and N5H6F1S1) and 10
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glycosite-containing peptides. Figure 2A showed the relative intensities of [pep+N3H] ions under
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Analytical Chemistry
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four different HCD fragmentation energies (10%, 20%, 30% and 40%). To offer comparison
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among different spectrums, the relative intensity of [pep+N3H] ion in each spectrum was
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normalized separately to the ion with the highest intensity in the spectrum. Based on the results,
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20% HCD energy yielded the highest intensity of [pep+N3H] ion with an average of 21.4±12.8%,
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followed by 30% energy (5.6±3.4% intensity), 10% (0.8±0.4% intensity) and the last 40% (1.8%
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intensity with only one spectrum). Notably, the relative intensities of Y ions varied among different
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glycopeptides, one of the reasons might be the effects of the glycan fragmentation from both glycan
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structures and peptide sequences. Despite of that, it is obvious that 20% HCD generated the highest
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relative intensity of [pep+N3H] ions in order to detect the bisecting N-glycosylation. Furthermore,
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20% HCD energy is optimal for the intensities of Y2 [pep+HexNAc2], [pep+HexNAc2Hex1] ions
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as well (Data not shown).
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To evaluate this method for analyzing complex samples, we further analyzed the intact
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glycopeptides with bisecting N-glycans in kidney tissue from rats. The intact glycopeptides were
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enriched from renal tissues of rats using Oasis MAX SPE column and analyzed by triplicate LC-
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MS/MS. Two fragmentation energies (20% and 33% HCD) were used for the analysis, in which
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33% HCD energy generates b+ and y+ ions for peptide identification while 20% HCD was used
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for the determination of bisecting N-glycans. When the peptide sequences and glycan compositions
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of intact glycopeptides were identified using GPQuest, a cutoff of 1% FDR (false discovery rate)
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were used to exclude false-positive results. By using the [pep+N3H] ion as a characteristic ion for
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bisecting glycan recognition, a total of 745 oxonium-containing PSMs were identified as
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glycopeptides with bisecting glycans attached (Table S1). The FDR for this method was evaluated
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using decoy spectra in which a random mass ranging from 3-20 was added to the mass of the
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original [pep+N3H] ion. After both theoretical target and decoy spectra were competitively 5
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matched against the experimental spectrum, the FDR of this result was estimated to be 1.6%.
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Furthermore, the presence of O-GlcNAc together with N-glycan on the same peptide might
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produce the same characteristic ion [pep+N3H] in the spectrum, which fortunately occurs very
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rarely. In such case, extra caution needs to be taken and the absence of another characteristic ion
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[pep+HexNAc3] may be used to exclude the possible false identification.
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To analyze the impact of core fucosylation (CF) modification on the fragmentation of [pep+N3H]
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ion, we analyzed the intensities of [pep+N3H] ion in bisecting N-glycans with and without CF
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modification (Figure 2B). As it is known, CF often occurs together with bisecting N-glycans,
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which both modify the core of the N-glycans16. With the CF modifies the GlcNAc linked to the
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Asparagine within N-X-S/T motif, it is possible that the intensity of [pep+N3H] ion might be
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influenced by the generation of the other characteristic ion [pep+N3HF]. Surprisingly, the relative
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intensities of [pep+N3H] ion from bisecting N-glycans were only decreased from 17.3±11.2% to
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13.5±7.4% when the CF modification occurs. Therefore, [pep+N3H] ion as a characteristic Y ion
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can still be used when analyzing the CF modified-bisecting N-glycans.
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We next evaluated whether the [pep+N3HF] ion would enhance the recognition of bisecting N-
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glycosylation with CF modification compare to the [pep+N3H] ion. First, we compared the relative
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intensities of the [pep+N3H] ion to the [pep+N3HF] ion, which was produced solely by the CF-
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modified bisecting N-glycans. As shown in Figure 2C, the intensities of [pep+N3HF] ion were
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significantly higher than that of [pep+N3H] ion. This result suggested that [pep+N3HF] ion has a
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better performance than [pep+N3H] ion for the identification of bisecting N-glycans with CF-
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modification. To this end, we analyzed all spectra (805 PSMs) with [pep+N3H] ion and
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[pep+N3HF] ion (Figure 2D). The use of [pep+N3HF] ion added around 21.9% more spectra for
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the identification of glycopeptides with CF-modified bisecting N-glycans, which accounted for ~8% 6
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Analytical Chemistry
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more spectra for the identification of glycopeptides with all bisecting N-glycans. This proved that
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the use of both [pep+N3H] and [pep+N3HF] ions indeed enhanced the capability of our approach.
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To further address the rigor of the method, a control experiment was performed with the
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Schneider 2 cells (S2 cells) from Drosophila melanogaster, a widely used model insect. Since
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complex N-linked glycans are generally considered to be absent in insect cells, the Drosophila S2
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cells should contain no bisecting N-glycans and therefore serve as an optimal sample for negative
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control17. With the peptides extracted from GPQuest software (FDR
