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Variation of Glycosylation Patterns Revealed by PGC‐LC‐ and Gated‐TIMS‐EED Tandem Mass Spectrometry
Author(s) -
Wei Juan,
Tang Yang,
Zaia Joseph,
Hong Pengyu,
Costello Catherine E.,
Lin Cheng
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.08858
Subject(s) - chemistry , glycan , mass spectrometry , glycomics , tandem mass spectrometry , electron transfer dissociation , electron capture dissociation , fourier transform ion cyclotron resonance , chromatography , glycosylation , ion mobility spectrometry , analytical chemistry (journal) , biochemistry , glycoprotein
Glycans are the most abundant biopolymers found in nature; they play crucial roles in a wide range of biological activities. Glycan biosynthesis does not follow genetic blueprints, and the resultant glycome is often a mixture of glycoforms with varying compositions, branching patterns, linkages, and stereochemical configurations. Subtle changes in the structure of a glycan can have a profound impact on its biological functions. Recently, we have demonstrated the potential of porous graphitic carbon liquid chromatography electronic excitation dissociation tandem mass spectrometry (PGC‐LC EED MS/MS) and gated‐trapped ion mobility spectrometry (TIMS) negative electron transfer dissociation (NETD) MS/MS in characterizing glycan mixtures, including the separation and identification of isomeric structures. Here, by studying N‐ glycans released from different batches of glycoproteins, the glycoform variations at the isomer level were observed. The detailed structure of each isomer could be determined by EED MS/MS and their relative quantification may be achieved by high‐resolution LC or ion mobility separation. Methods The released N‐ linked glycans were reduced and permethylated before analysis to improve the ionization efficiency and to facilitate spectral interpretation. All experiments were carried out on a 12T solariX TM Fourier‐transform ion cyclotron resonance mass spectrometer equipped with a prototype TIMS device (Bruker Daltonics) or a nanoACQUITY UPLC system (Waters). Results Ribonuclease B (RNase B) is the glycosylated form of RNase A. With the attachment of a glycan at Asn‐34, the glycosylated ribonuclease showed an increased structural flexibility in the RNA binding region and a much higher activity on double‐stranded RNAs. Previously, application of PGC‐EED MS/MS to the analysis of N ‐glycans released from RNase B showed the presence of 18 high‐mannose structures. Here, by comparing the PGC‐LC MS/MS results from two batches of RNase B purchased from different manufacturers, we observed variations in the abundance of each glycoform. Additionally, we have implemented EED MS/MS with gated‐TIMS separation, and showed its ability to resolve and identify isomeric structures. Using gated‐TIMS EED MS/MS, we were able to determine the batch‐to‐batch variation of chicken ovalbumin N ‐glycans along with the conformational information. Conclusions The PGC‐ and gated‐TIMS‐ EED MS/MS approaches showed advanced performance in separation, characterization, and relative quantification of isomeric glycans, as well as in applications to determine the batch‐to‐batch glycoform variations of glycoproteins. These approaches hold great potential for comprehensive and confident glycan characterization. Support or Funding Information This research is supported by the NIH grants P41 GM104603, 703 R21 GM122635, U01CA221234, R01 GM132675, and S10 704 RR025082.