Stabilization of Sialic Acids in N-linked Oligosaccharides and Gangliosides for Analysis by Positive Ion Matrix‐assisted Laser Desorption/Ionization Mass Spectrometry

Abstract

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry of oligosaccharides and gangliosides normally causes loss of sialic acid, particularly when alpha-cyano-4-hydroxycinnamic acid is used as the matrix. In addition, the potential signal is split because both positive and, to a greater extent, negative ions are formed while signals are frequently complicated as the result of partial alkali-salt formation. In order to stabilize the sialic acid moieties under MALDI conditions and to divert all of the signal into the positive-ion mode, a method involving their conversion into methyl esters has been developed. The method is relatively rapid and produces strong positive-ion signals from N-linked oligosaccharides containing sialic acid and from gangliosides. The latter compounds are stable, even in the presence of alpha-cyano-4-hydroxycinnamic acid. They give abundant molecular (MNa+) ions, but with sufficient residual in-source fragmentation to allow the sequence of the sugar chain to be determined. The sialic acid residue is stable after methylation, irrespective of its linkage to the parent molecule.

Analysis of protein glycosylation by mass spectrometry

“…Neutral glycans yield intense signals in the positive ion mode corresponding to sodium-cationized molecular species [ 23 . Permethylation of glycans using the method described by Ciucanu and Kerek also stabilizes the sialic acid residues 24 .…”

Targeted Proteo-Glycomics Analysis of Sialyl Lewis X Antigen Expressing Glycoproteins Secreted by Human Hepatoma Cell Line

“…Several mass spectrometric approaches for analysis of glycan structures have been reported. [11][12][13][14][15][16][17]34,35 We analyzed N-glycans of these glycoproteins using MALDI-TOFMS in combination with exoglycosidase digestion. The results indicated that increases in α1-3 fucosylated, poly-fucosylated, and highbranched N-glycans occurred not only on transferrin but also on α1-antitrypsin, α2-HS-glycoprotein, and β2-glycoprotein.…”

MALDI-MS/MS with Traveling Wave Ion Mobility for the Structural Analysis ofN-Linked Glycans

“…Sialylated glycans from bovine fetuin and thyroglobulin appeared to lose much less of their sialic acid than when examined with the MALDI-TOF system; spectra were compared with those of stable permethylated samples (spectra not shown). However, even if desialylation is a problem, previous work has shown that sialic acid loss can be prevented by derivatization of the carboxy group of the sialic acids by methyl ester [33,34] or amide formation [35,61] or by selective formation of lactones from α2→3-linked sialic acids [34] with retention of the free hydroxyl groups that are necessary for production of the informative negative ion CID spectra.…”

“…The spectra of monosialylated glycans, in particular, retain many of the diagnostic ions (data not shown but see reference [64]). However, these glycans can be neutralized by derivatization [33,61], as mentioned above and, in some cases, the derivatization reaction can be exploited to provide linkage information for the sialic acids [34].…”

“…Most of the work in this area has been performed with electrospray ionization which has the disadvantages of multiple charging, discrimination against the larger glycans and formation of in-source fragments leading to rather complex spectra. MALDI is a more satisfactory technique for obtaining glycan profiles of neutral carbohydrates although sialylated compounds tend to eliminate sialic acids under MALDI conditions unless derivatized by, for example, permethylation, methyl ester [33,34] or amide [35] formation. Neutral glycans, however, do not easily form ions by MALDI in negative ion mode with the normal matrices such as 2,5-dihydroxybenzoic acid (DHB) but can be induced to do so by use of matrices such as nor-harmane [36] or by adding suitable anions to the matrix [37][38][39][40][41][42][43].…”