Derivatization of sialic acids for stabilization in matrix‐assisted laser desorption/ionization mass spectrometry and concomitant differentiation of α(2 → 3)‐ and α(2 → 6)‐isomers

Abstract

Sialylated carbohydrates usually decompose by loss of sialic acid when ionized by matrix-assisted laser desorption/ionization (MALDI) as the result of the labile carboxylic proton. Stabilization has previously been achieved by forming methyl esters with methyl iodide, a procedure that eliminates the labile proton. In this paper, we describe an alternative procedure for methyl ester formation that provides information on the sialic acid linkage directly from the MALDI spectrum. The sugars were desalted, dissolved in methanol, and treated with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM). After removal of the solvent, the products were transferred directly to the MALDI target and examined from 2,5-dihydroxybenzoic acid. Small amounts of N-glycans derived from biological sources benefited from an additional clean-up stage involving Nafion 117. alpha(2 --> 6)-Linked sialic acid produced only methyl esters whereas alpha(2 --> 3)-linked sialic acids were converted into their lactones providing a 32 Da difference in mass. Negative ion collision-induced decomposition (CID) mass spectra of these neutralized glycans provided information, in many cases, on the antenna of N-linked glycans to which the variously linked sialic acids were attached. The method was applied to N-linked glycans released from bovine fetuin and porcine thyroglobulin.

Sensitive and Structure-Informative <i>N</i>-Glycosylation Analysis by MALDI-MS; Ionization, Fragmentation, and Derivatization

“…Originally, Wheeler et al introduced linkage-speci c methylesteri cation, which is based on di erences in the reactivity of carboxyl groups on sialic acids. 67) Under the derivatization conditions, α2,6-linked sialic acids are converted to esters, whereas α2,3-linked sialic acids form lactones by intramolecular dehydration. e resulting mass di erence obtained from the mass spectra allows the linkage-speci c di erentiation of the types of sialic acids.…”

“…In fact, Wheeler et al pointed out that lactones can be completely degraded within 50 h by simply dissolving them in water. 67) is instability can limit the downstream chemical and/or enzymatic treatments. 70,71) As pointed out by other researchers, lactone forms cannot survive enzymatic digestion, resulting in partial de-lactonization.…”

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].…”