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Complexity and Diversity of the Mammalian Sialome Revealed by Nidovirus Virolectins
Abstract
based on crystal structures (α-Neu5,9Ac 2 , α-Neu5,7,9Ac 3 and α-Neu4,5,9Ac 3 PDB ID: 3CL5 (Zeng et al., 2008) ; α-Neu4,5Ac 2 , PDB ID: 4C7W (Langereis et al., 2012) ; α-Neu5Gc, PDB ID:
2WYK (Byres et al., 2008) , modified with PYMOL to add or delete O-acetyl moieties. In the Kekulé structures, pertinent Sia modifications are indicated in red. (C) GLC-EIMS analysis of Sia constituents of BSM. Total-ion-current chromatograms of sialic acids released from BSM, using either propionic-acid (top) or Arthrobacter ureafaciens sialidase (bottom), and converted into volatile pertrimethylsilylated derivatives (Kamerling and Gerwig, 2006) . For explanation of the numbered peaks, see Table S2 . (Zeng et al., 2008 , Langereis et al., 2009 Langereis et al., 2012) . N.A., not analyzed.
chromatography-electron ionization mass spectrometry (GLC-EIMS). The Sia composition of bovine submaxillary mucin (BSM) as analyzed by GLC-EIMS has been reported (Reuter et al., 1983) . However, as O-Ac groups are unstable and subject to C7-to-C9 migration (Kamerling et al., 1987) , large differences may exist in O-Ac-Sia content among different batches of BSM.
Moreover, in order to perform GLC-EIMS, Sias have to be released from the penultimate sugar residues, the classical procedure for which is incubation with 2 M propionic acid at 80°C (Kamerling and Gerwig, 2006) . These conditions are known to have an effect on the labile Oacetyl groups in terms of loss and migration. For our analyses, it was crucial that the O-Ac-Sia composition as measured by GLC-EIMS reliably reflected that of native BSM. We therefore established an alternative protocol to release the Sias enzymatically, with all experimental procedures performed under conditions that would minimize O-acetyl release/migration. BSM (500 µg) was dissolved in 500 µl PBS, pH 6.5 and treated with 50 mU of Arthrobacter ureafaciens sialidase (AuS; Sigma) for 4 h at 37°C (note that reactions were performed at mildly acidic pH to prevent migration C7-O-acetyl moieties to C9; (Kamerling et al., 1987) . For comparison, the same amount of BSM was dissolved in 500 µl 2 M propionic acid and incubated at 80°C for 4 h. Sia samples were lyophilized and trimethylsilylated with a mixture of trimethylchlorosilane : hexamethyldisilazane : pyridine (1:1:5) for 30 min at RT. Derivatized samples were separated by GLC with a temperature program of 25 min at 220°C, followed by a gradient from 220°C to 300°C at 6°C/min, and finally for 6 min at 300°C using a Fisons Instruments GC8060 gas chromatograph equipped with an AT-1 column (30 m x 0.25 µm,
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