Quantitative Derivatization of Sialic Acids for the Detection of Sialoglycans by MALDI MS

Abstract

Recently, glycans have been recognized as valuable biomarkers for various disease states. In particular, sialoglycans, which have sialic acids at their terminal end, are likely to have relevance to diseases such as cancer and inflammation. Mass spectrometry (MS) has become an indispensable tool for biomarker discovery. However, matrix-assisted laser desorption ionization (MALDI) MS of sialoglycans normally causes loss of sialic acid. Methylesterification or amidation of carboxyl functionality in sialic acid has been reported to suppress the loss of sialic acids. We found that the modifications of alpha2,3-linked sialic acids proceed less efficiently than those at alpha2,6-linkages. Furthermore, the modifications of the alpha2,3-linked sialic acids are incomplete. This variability in the extent of derivatization presents a major problem in terms of glycan biomarker discovery using MALDI MS. In this study, we developed a novel amidation using acetohydrazide which can completely modify both types of linkages of sialoglycans. With the use of this method, we demonstrate MS profiling of N-linked glycans released from a bovine fetuin which is rich in alpha2,3-linked sialic acids.

Identification and characterization of sulfated glycoproteins from small cell lung carcinoma cells assisted by management of molecular charges

“…Briefly, the glycopeptide mixture was subjected to sialidase digestion followed by carboxypeptidase B digestion and finally acetohydrazidation as follows. The mixture was dissolved in 250 μL of 2.5 M acetohydrazide, and the pH of the solution was adjusted to 2.5 by adding a small amount of 1 M HCl [29]. Five microliters of 4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was added to the solution, and the mixture was incubated at room temperature for 1 h. This addition and the incubation steps were then repeated an additional 3 times.…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2007–2008

“…3-linked sialic acids, Toyoda et al (2008) have investigated alternative methods of derivatizing these groups and have produced a method using ANALYSIS OF CARBOHYDRATES AND GLYCOCONJUGATES & acetohydrazide (24) derivatization. The reaction appeared to be quantitative with both a2 !…”

Solid-phase methylamidation for sialoglycomics by MALDI-MS

“…The incomplete conversions may be due to the steric hindrance effects of α (2,3)-linked sialic acids. Similar phenomena would be occurred in derivatization sialic acid glycans by methylesterification and amidation which could not fully derivatize sialoglycans containing α (2,3)-linked sialic acids as previously reported [22]. With the active carboxylate group of the reagent (7-azabenzotriazol-1-yloxy) tripyrroldinophosphoniumhexaflorophosphate (PyAOP), nonspecific methylamidation of both α (2,3)-and α (2,6)-linkage types of sialic acids could be realized.…”