Differentiation of Sialyl Linkages Using a Combination of Alkyl Esterification and Phenylhydrazine Derivatization: Application for N-Glycan Profiling in the Sera of Patients with Lung Cancer

Abstract

Alterations in oligosaccharides and types of sialic acid (SA) attachments have been associated with different pathological states. Matrix-assisted laser desorption mass spectrometry (MS) is commonly used for glycosylation studies. However, native sialylated glycans are suppressed or not detected during MS experiments. Consequently, different approaches have been employed to neutralize the negative charge of the carboxyl group. In this study, we present the advantage of phenylhydrazine (PHN) labeling for the detection and efficient discrimination of SA linkages when this derivatization follows alkyl esterification. As expected, PHN-labeled sialylated oligosaccharides with the 2,6linkage type can be easily recognized according to the additional shift in mass corresponding to the presence of a methyl or ethyl group. Surprisingly, oligosaccharides with the 2,3-linked SA residue instead of a lactone were detected carrying the second PHN unit. This was beneficial as no further processing after esterification was needed to stabilize the lactone form. Moreover, during tandem mass experiments, all modified glycans produced favorable fragmentation patterns with a coherent recognition of SA linkages. Although both types of esterification, herein called the EST−PHN approach, provided comparable results, methylation exhibited marginally higher linkage specificity than ethyl esterification. The simplicity and effectiveness of the methodology are demonstrated on the model compound, sialyllactose, and its applicability for biological studies is presented on N-glycan profiling in the sera of lung cancer patients.

N-glycan profiling of tissue samples to aid breast cancer subtyping

“…However, it must be recognized that this approach is not optimal for distinguishing all structural isomers, such as α-2,3- from α-2,6-linkages of sialic acids. These structural entities that may be functionally important 39 , 40 are being addressed by the development of alternative methodologies 41 – 43 and have also been applied to patient samples 44 , 45 .…”

Targeting protein glycosylation to regulate inflammation in the respiratory tract: novel diagnostic and therapeutic candidates for chronic respiratory diseases

“…Glycan derivatization can be achieved through derivation of the reducing end, hydroxyl group, and carboxyl group ( 201 ). For example, phenylhydrazine labeling (a reducing end derivation method) has been used for structural studies of fucosylated N-glycans by MALDI-MS in positive ion mode ( 208 ), and for efficient detection and discrimination of SA linkages when following alkyl esterification ( 209 ). MS-based systems commonly used for glycomic characterization include MALDI, electrospray ionization (ESI), capillary electrophoresis (CE), gas chromatography (GC) and liquid chromatography (LC) coupled to MS ( 143 ).…”

Two‐Dimensional Tandem Mass Spectrometry for Biopolymer Structural Analysis

“…Derivatization reactions have been used in mass spectrometry to alter the structure of certain analytes in such a way as to provide more effective qualitative and quantitative analysis. [10] To explore the use of 2D MS/MS in conjunction with derivatization, the N-terminus of YGGFL was acetylated so that N-terminus-carrying ions (a/b/c) could be identified and separated from others, e.g. y-ions.…”