Abstract
Negative ion collision-induced dissociation (CID) of underivatized N-glycans has proved to be a simple, yet powerful method for their structural determination. Recently, we have identified a series of such structures with GalNAc rather than the more common galactose capping the antennae of hybrid and complex glycans. As part of a series of publications describing the negative ion fragmentation of different types of N-glycan, this paper describes their CID spectra and estimated nitrogen cross sections recorded by travelling wave ion mobility mass spectrometry (TWIMS). Most of the glycans were derived from the recombinant glycoproteins gp120 and gp41 from the human immunodeficiency virus (HIV), recombinantly derived from human embryonic kidney (HEK 293T) cells. Twenty-six GalNAc-capped hybrid and complex N-glycans were identified by a combination of TWIMS, negative ion CID, and exoglycosidase digestions. They were present as the neutral glycans and their sulfated and α2→3-linked sialylated analogues. Overall, negative ion fragmentation of glycans generates fingerprints that reveal their structural identity.
Highlights
Fragmentation by collision-induced dissociation of negative ions from N-linked glycans (those attached to proteins at Published in the topical collection Analytical Characterization of Viruses with guest editor Joseph Zaia.asparagine in an Asn-Aaa-Ser(Thr) consensus sequence, where Aaa is any amino acid except proline) has been shown to provide a wealth of diagnostic cross-ring fragments
We have been systematically examining the negative ion fragmentation of different types of N-glycans [5,6,7,8,9,10,11] and in this report present negative ion collision-induced dissociation (CID) spectra and ion mobility properties of glycans containing N-acetylgalactosamine (GalNAc) terminating their antennae and their identification in the human immunodeficiency virus (HIV) envelope glycoprotein (Env) BG505 SOSIP.664 trimers expressed in Human embryonic kidney (HEK) 293T cells
Glycans were released from BG505 SOSIP.664 trimers that were transiently expressed in HEK 293T cells (Thermo Fisher Scientific, Waltham, MA) and purified by 2G12-affinity chromatography followed by size-exclusion chromatography, as previously described [26, 27]
Summary
Fragmentation by collision-induced dissociation of negative ions from N-linked glycans (those attached to proteins at Published in the topical collection Analytical Characterization of Viruses with guest editor Joseph Zaia.asparagine in an Asn-Aaa-Ser(Thr) consensus sequence, where Aaa is any amino acid except proline) has been shown to provide a wealth of diagnostic cross-ring fragments. Some of these structural features are difficult to determine by traditional methods such as exoglycosidase sequencing as these enzymatic reactions are often incomplete plus they are often time-consuming and expensive For acidic glycans, those with sialic acid can be converted to neutral compounds by methyl ester [2] or amide formation and, depending on the method used, the resulting spectra provide information on sialic acid linkage by utilizing their different reactivities to the derivatizing reagents such that the linkage is revealed by mass difference [3, 4]. We have been systematically examining the negative ion fragmentation of different types of N-glycans [5,6,7,8,9,10,11] and in this report present negative ion collision-induced dissociation (CID) spectra and ion mobility properties of glycans containing N-acetylgalactosamine (GalNAc) terminating their antennae and their identification in the human immunodeficiency virus (HIV) envelope glycoprotein (Env) BG505 SOSIP.664 trimers expressed in HEK 293T cells
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