Linkage-specific characterization of sialylated n-glycans from human plasma by lc-ms

Session: 
S3.3 Glycomics
Code: 
OL3.3.4
Location (hall): 
Galactose
Start/end time: 
Monday, July 1, 2019 - 18:00 to 18:15
Alan
Moran

Alan Moran1,2, Richard Gardner2, Jennifer Hendel2, Daniel I.R. Spencer2

1Leiden University Medical Center, Leiden, The Netherlands, 2Ludger Ltd., Abingdon, United Kingdom

Introduction:
The developing era of precision medicine requires novel disease biomarkers that have molecular characteristics with predictive and/or prognostic value. For example, molecules such as α(2,3)- and α(2,6)- linked sialic acid on N-glycans have been implicated in studies investigating malignant transformation [1]. In these cases, aberrant sialylation can be analysed using several methods including liquid chromatography-mass spectrometry (LC-MS) and MALDI-MS [2]. Various sialic acid chemical modification techniques have been applied in MALDI-MS analyses in order to allow linkage-specific characterization [3]. In comparison, sialic acid linkage differentiation using LC is often based on retention times and/or sequential exoglycosidase digestions [4]. However, few LC-MS methods exist that allow linkage-specific characterization of sialic acids [5].

Methods:
A novel experimental approach for sialic acid linkage differentiation will be presented that involves the selective chemical derivatization of sialylated N-glycans followed by analysis using LC-MS. First, PNGase-F released N-glycans from human plasma were fluorescently labelled using procainamide, followed by removal of excess labelling reagent using a hydrophilic interaction chromatography (HILIC)-based technique (GHP) [6]. Next, ethyl esterification and amidation of sialic acids was carried out with a subsequent GHP membrane clean-up [3]. Finally, modified N-glycans were analysed by HILIC-MS using a (Waters) BEH-glycan column.

Results:
There were prominent differences in the retention times associated with derivatized vs. non-derivatized N-glycans (figure 1). In general, derivatization resulted in overall earlier elution of the sialylated glycans whereby glycans with α(2,6)-linkages were most effected. The final assignment of derivatized sialylated structures was carried out based on three main features: retention times specific to sialic acid linkage, parent ion mass and specific molecular ions. As a result, a more in-depth characterization of human plasma N-glycans could be made. Thus, this study supports the use of chemical modification followed by LC-MS analysis as a platform to characterise α2,3- and α2,6-sialylated glycan isomers. Importantly, this may have implications for precision medicine whereby abnormal changes in these monosaccharides may represent a potential new class of disease biomarker.

Figure 1: Extracted ion chromatograms of procainamide labelled A2G2S2 N-glycan from human plasma. LC-MS analysis was performed using hydrophilic interaction liquid chromatography mass spectrometry (HILIC-MS) with a (Waters) BEH-glycan column. Top: Underivatized N-glycan is shown. Bottom: Display of derivatized N-glycan(s) for α(2,6) α(2,6) (left), α(2,3)α(2,6) (middle), and α(2,3)α(2,3) (right).

References: 
  1. Zhang Z.; Wuhrer M.; Holst S. Serum sialylation changes in cancer. Glycoconjugate journal. 2018 Apr 21:1-22.
  2. Nie H.; Li Y.; Sun XL. Recent advances in sialic acid-focused glycomics. Journal of proteomics. 2012 Jun 18;75(11):3098-112.
  3. Reiding KR.; Blank D.; Kuijper DM.; Deelder AM.; Wuhrer M. High-throughput profiling of protein N-glycosylation by MALDI-TOF-MS employing linkage-specific sialic acid esterification. Analytical chemistry. 2014 May 28;86(12):5784-93.
  4. Houel S.; Hilliard M.; Yu YQ.; McLoughlin N.; Martin SM.; Rudd PM.; Williams JP.; Chen W. N-and O-glycosylation analysis of etanercept using liquid chromatography and quadrupole time-of-flight mass spectrometry equipped with electron-transfer dissociation functionality. Analytical chemistry. 2013 Dec 18;86(1):576-84.
  5. Tousi F.; Bones J.; Hancock WS.; Hincapie M. Differential chemical derivatization integrated with chromatographic separation for analysis of isomeric sialylated N-glycans: a nano-hydrophilic interaction liquid chromatography-MS platform. Analytical chemistry. 2013 Aug 19;85(17):8421-8.
  6. Kozak RP.; Tortosa CB.; Fernandes DL.; Spencer DI. Comparison of procainamide and 2-aminobenzamide labeling for profiling and identification of glycans by liquid chromatography with fluorescence detection coupled to electrospray ionization–mass spectrometry. Analytical biochemistry. 2015 Oct 1; 486:38-40.

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