The effect of serum N-glycan derivatization in reflectron positive MALDI mass spectra

Authors: Zuzana Pakanová 1    Marek Nemčovič 1    Filip Pančík 1    Barbara Siváková 1    Zuzana Brnoliaková 2    Peter Baráth 1   
1 Slovak Academy of Sciences, Institute of Chemistry, Bratislava    2 Slovak Academy of Sciences, Centre of Experimental Medicine, Bratislava   
Year: 2020
Section: Utilization of instrumental methods in the analysis of biologically important substances
Abstract No.: 1961
ISBN: 978-80-972360-6-9

Altered serum N-glycoprofile was observed in many pathological conditions such as diabetes [1], cancer [2] and metabolic disorders [3]. N-glycans, cotranslationally added to proteins, play an essential role in physiological processes. Thus, complex knowledge about their structures can lead to better understanding of molecular basis of various diseases and they serve as potential biomarkers. In our study we analysed the representative serum pool of male healthy individuals to determine the effect of N-glycan derivatization on the obtained quality and intensity of signals in reflectron positive ion mode.

After the denaturation of serum proteins, N-glycans were released by peptide-N-glycosidase F and isolated by PGC-SPE. To acquire the MALDI TOF mass spectra of free serum N-glycans, sample was subjected to C18-reverse phase SPE to remove the contaminants. The first derivatization approach was based on fluorescent labelling of reducing ends by 2-aminopyridine and the second derivatization alternative included the permethylation of all accessible hydroxy-groups in glycan molecules. Obtained MS and representative MS/MS spectra of free, permethylated and 2-PA labelled N-glycans are compared and discussed.

Each derivatization of released N-glycans presents both advantages and disadvantages and should be considered according to the expected structures. Analysis of free N-glycans is an appropriate approach when no sialylated glycans are expected in the sample. Both permethylation and fluorescent labelling involve extra preparation steps that can lead to the loss of sample and are methodologically demanding. However, such derivatization strategies enable increased intensities of acquired signals and improved spectra quality. In the fragmentation pattern, derivatization has no effect on the typical major glycosidic bond cleavage yielding sequence but not linkage information.

This work was supported by Ministry of Health of the Slovak Republic under the project registration number 2019/7-CHÚSAV-4; VEGA 2/0130/18 and APPV-18-0336.
[1] RUDMAN, N., GORNIK, O., LAUC, G. (2019), FEBS Letters, 593: p. 1598-1615, Altered Nglycosylation profiles as potential biomarkers and drug targets in diabetes.
[2] LIU, S., HUANGA, Z., ZHANG, Q., et al. (2020), Biochimica et Biophysica Acta - General Subjects, 1864 (3), Profiling of isomer-specific IgG N-glycosylation in cohort of Chinese colorectal cancer patients.
[3] FREEZE, H. (2019), Annals of Translational Medicine, 7 (Suppl. 6), S176. Improving biochemical markers for disorders of N-glycosylation.