Sialic acid mass spectrometry

Great progress in our knowledge about sialic acids was made with the aid of gas-liquid chromatography (g.l.c.), mass spectrometry (m.s.), and n.m.r. spectroscopy. Structural analysis of the many different N- andO-acylated Neu derivatives occurring in Nature will be described in Sections IV,5 and 6. 

Although the different methods for the analysis of sialic acids thus far described give some insight into the structure of these compounds, mass spectrometry permits unambiguous determination of the chain length and the number, type, and position of N- or O-acyl substituents in Neu derivatives. Thus, a variety of new and rare sialic acids have been discovered in the past few years, and the presence of known, mainly O-acetylated, sialic ac ids has been established in a variety of biological materials.34-37,62 85,94,95-97-96-10 -107-147-149... 

Mass Units of Characteristic Fragment-ions A-G Used for the Identification of Natural, N,O-Acylated Sialic Acids, and of the C7 and C8 Analogs of Neu5Ac and Neu5Gc as their Methyl Esters, Trimethylsilyl Ethers, by Mass Spectrometry"... 

Several alternative methods for the determination of sialic acid in body fluids and tissues have been described. Most of these methods make use of the classic periodate-TBA assay in combination with purification using HPLC [13]. Another method makes use of fluorometric HPLC of sialic acids after derivatization with a fluorogenic compound [9]. The most promising new method for the determination of free sialic acid in urine (and probably also other body fluids and tissues) is the HPLC-tandem mass spectrometry method [19]. This method is rapid, accurate, and sensitive, and is more robust than earlier methods. The only disadvantage is the expensive equipment that is required, which makes it only economical for specialized metabolic laboratories. Since this equipment is used for many different metabolic assays, the investment is certainly warranted, and nowadays almost essential for any metabolic laboratory. 

Valianpour F, Abeling NG, Duran M, Huijmans JG, Kulik W (2004) Quantification of free sialic acid in urine by HPLC-electrospray tandem mass spectrometry a tool for the diagnosis of sialic acid storage disease. Clin Chem 50 403-409... 

Powell AK, Harvey DJ (1996) Stabilization of sialic acids in N-linked oligosaccharides and gangliosides for analysis by positive ion matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun Mass Spectrom 10 1027-1032... 

Mass spectrometry has been used successfully to establish the structure of sialic acids, and the type of their bonds in the oligosaccharide chain. For this purpose, the fragmentation of various derivatives containing both the cyclic and the acyclic forms of sialic acid has been comprehensively studied. The methyl esters of sialic acid methyl ketosides, obtained as a... 

Ten sialic-acid-containing oligosaccharides and four neutral oligosaccharides have been purified from the major glycoprotein fraction of hog, submaxillary-gland mucin. H-N.m.r. spectroscopy at 360 MHz, in combination with methylation analysis and mass spectrometry, allowed the determination of their structures, which are partial structures of a pentasaccharide (see Fig. 42) having blood-group A activity.221... 

G. P. Smirnova, N. V. Chekareva, O. S. Chizhov, B. M. Zolotarev, and N. K. Kochetkov, Mass spectrometry of sialic acids Peracetylated methyl esters of 5-acylamino-3,5-dideoxy-nononic and -heptonic acids, Carbohydr. Res., 59 (1977) 235-239. 

The first and the second NCL gave the desired glycosylpolypeptide and subsequent oxidative folding yielded MCP-3. The glycosylpolypeptide thus obtained was treated with 50mM NaOH solution for lOmin to remove the Bn groups from the sialic acid residues to afford MCP-3, which was characterized by mass spectrometry, HPLC, circular dichroism (CD), disulfide mapping, and enzyme-linked immunosorbent assay. 

Y. Miura, Y. Shinohara, J.-i. Furukawa, N. Nagahori, and S.-I. Nishimura, Rapid and simple solid-phase esterification of sialic acid residues for quantitative glycomics by mass spectrometry, Chem. Eur. J., 13 (2007) 4797 1804. 

If possible, typical biological sources have been indicated. In the case of a sialic acid structure being proven by mass spectrometry and/or NMR spectroscopy, reference numbers refer in general to such studies or to review articles including this sialic acid. 

Thus mass spectrometry showed the GPI was heterogeneous with at least five separate species and its composition was consistent with known GPI structures but with sialic acid as a novel component. The heterogeneity was confirmed by various separative techniques including capillary electrophoresis, Dionex high performance anion exchange chromatography (HPAEC), and reverse phase HPLC at pH 7. Cleavage... 

Figure 44 Strategy for identifying the binding partners of sialosides. Metabolic engineering can be used to replace sialic acids with their photocross-linking counterparts. Modified glycoconjugates can be subjected to in situ irradiation to form cross-links with their binding partners. The covalently bound complexes can be isolated and analyzed by immunochemical or mass spectrometry methods.

The use of fluorinated sialic acid analogues to trap intermediates opens the concern that the reaction mechanism has been altered. This concern was overcome in recent studies wherein classical ping-pong kinetics were observed for TcTS, consistent with the existence of a covalent intermediate. In addition, incubation of the acid/base mutant D59A TcTS with an activated substrate,/)-nitrophenyl a-sialoside (PNP-SA), resulted in a pre-steady-state burst of PNP release whose magnitude corresponded well with the amount of TcTS used. Further, when PNP-SA was incubated with either D59A TcTS or wild-type TcTS, the accumulation of the sialyl-enzyme intermediate was clearly observed by mass spectrometry. 

Despite a level of sensitivity about 100 times lower than that obtained by CE-UV, inductively coupled plasma mass spectrometry (ICP-MS) coupled to CZE has been shown to separate and distinguish between isoforms containing 3,4, and 5 sialic acid residues in standard Tf samples [133]. 

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