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Presentation Type: Oral presentation
Session Topic: Carbohydrates
Abstract Identification Number: 10838-6
Releasing N-glycan from Peptide N-terminus by N-terminal Succinylation Assisted
Enzymatic Deglycosylation
Yejing Weng1,2, Zhigang Sui1, Lihua Zhang1*, Yukui Zhang1
1Key
Lab of Separation Sciences for Analytical Chemistry, National Chromatographic R. & A. Center, Dalian Institute of
Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
2University
of Chinese Academy of Sciences, Beijing 100049, China
Duo to the important roles of glycoproteins in various biological processes, the global
glycoproteome analysis has been paid much attention. In common N-glycoproteome studies, the
advanced enrichment techniques, coupled with multidimensional chromatographic separation and highresolution mass spectrometry (MS), have dramatically enhanced the dynamic range and limit of
detection for N-glycosylation sites mapping. However, for peptides with glycosylated Asn at N-terminus
(PGANs), the amide bond between the N-linked glycan and the glycosylated Asn residue is difficult to
hydrolyze by PNGase F, making such sites extensively neglected in current glycoproteomic studies.
To address this problem, herein, we presented a facile strategy by incorporating succinylation at
the N-terminus of PGANs for improving the efficiency of enzymatic deglycosylation catalyzed by
PNGase F (Fig. 1). The N-glycopeptides from the tryptic digests of Ribonuclease B (RNase B) were
used to evaluate our proposed strategy. As shown in Fig. 2, the PGANs with the peptide sequence of
N#LTKDR can only be deglycosylated after the site-selective N-terminal succinylation. The mechanism
of succinylation assisted enzymatic deglycosylation was discussed, and introducing an amido linkage at
N-terminus of PGAN was proved to be crucial for increasing the stability of enzyme-substrate complex
and hence greatly accelerated the enzymatic deglycosylation catalyzed by PNGase F.
This strategy was further applied to the glycoproteome analysis of HeLa cell lysate. The low
percentage of identified PGANs (4.1%) in Route A was mainly attributed to the poor activity of PNGase
F for PGANs. As expected, more than 75% of the N-glycopeptides identified in Route B were PGANs,
indicating most PGANs were missed in our traditional glycoproteomic studies. Furthermore, to
demonstrate a general usefulness of the N-terminal succinylation assisted approach, the Nglycoproteome analysis of mouse brain was also performed (Fig. 3). The succinylation approach was
demonstrated had an advantage over the N-glycosylation mapping approach using two proteases (trypsin
and Glu-C). It is worth noting that the overlap between Route B (trypsin) and Route A (Glu-C) was only
7.6%, indicating these two approaches were complementary for profiling the N-glycosylation sites.
In conclusion, to facilitate PNGase F based deglycosylation, the PGANs were selectively separated
by two-step HILIC enrichment, then, a site-specific amide linkage was proposed to incorporate the Nterminus of PGANs by N-terminal succinylation. Such labeling has been proved to be crucial for the
identification of PGANs in glycoproteome samples, which improved the comprehensiveness of Nglycosylation sites mapping.
(The above work is unpublished)
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Fig. 1 Flowchart of N-glycopeptides profiling with combination of commonly applied protocol (Route
A) and our proposed protocol (Route B). SA: succinic anhydride.
Fig. 2 MALDI-TOF mass spectra of (a) N-glycopeptides enriched from RNase B tryptic digests, (b)
peptides treated by PNGase F, (c) PGANs enriched by HILIC, (d) PGANs labeled with SA, and (e)
deglycosylated and SA labeled PGANs. Man: mannose.
Fig. 3 Overlap of the three methods for mapping N-glycosylation sites