Characterization of glycopeptides from HIV-I_SF2 gp120 by liquid chromatography mass spectrometry

Abstract

Previously, we have characterized the HIV-I SF2 gp120 glycopeptides using matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) and nanospray electrospray ionization (ESI). Although we characterized 25 of 26 consensus glycosylation sites, we could not obtain any information about the extent of sialylation of the complex glycans. Sialylation is known to alter the biological activity of some glycoproteins, e.g., infectivity of some human and nonhuman primate lentiviruses is reduced when the envelope glycoproteins are extensively sialylated, and thus, characterization of the extent of sialylation of complex glycoproteins is of biological interest. Since neither MALDI/MS nor nanospray ESI provided much information about sialylation, probably because of suppression effects inherent in these techniques, we utilized online nanocapillary high performance liquid chromatography (nHPLC) with ESI/MS to characterize the sites and extent of sialylation on gp120. Eight of the known 26 consensus glycosylation sites of HIV-I SF2 gp120 were determined to be sialylated. Two of these sites were previously uncharacterized complex glycans. Thirteen high mannose sites were also determined. The heterogeneity of four of these sites had not been previously characterized. In addition, a peptide containing two consensus glycosylation sites, which had previously been determined to contain complex glycans, was also determined to be high mannose as well. (J Am Soc Mass Spectrom 2004, 15, 1545-1555

Macrophage-Derived Simian Immunodeficiency Virus Exhibits Enhanced Infectivity by Comparison with T-Cell-Derived Virus

“…Support for this idea comes from data revealing that the glycosylation patterns of the heavily glycosylated, mannose-rich gp120 (4,24) differ in a host cell-specific fashion (5,(10)(11)(12). We also find that several genes whose products degrade glycans are increased in macrophages.…”

“…We also find that several genes whose products degrade glycans are increased in macrophages. The fact that the majority of the glycoconjugates on gp120 contain high-mannose glycans (4,24) further strengthens the possibility that mannose residues on gp120 affect infectivity. On the other hand, differential glycosylation of numerous cellular proteins incorporated into virions in a cell type-dependent manner (1), as opposed to effects on viral proteins, could also change viral infectivity.…”

Mass spectrometric characterization of glycosylation of hepatitis C virus E2 envelope glycoprotein reveals extended microheterogeneity of N-glycans

“…For example, we have used a combination of MALDI-TOF and LC followed by nanoLC/MS/MS to characterize the glycan structures attached to the human immunodeficiency virus (HIV) gp120 glycoprotein, and high mannose glycans and hybrid glycans were found attached to the protein [32,45]. Although LC-MS analysis of released glycans may provide a detailed picture of the structure of the glycans derived from a protein or any complex protein mixture, information on the original attachment sites of the glycans and the underlying proteins is lost.…”

Comparison of HPLC/ESI-FTICR MS versus MALDI-TOF/TOF MS for glycopeptide analysis of a highly glycosylated HIV envelope glycoprotein

“…For each run, 20 L of the tryptic digest was injected onto a C18 column (150 ϫ 4.6 mm, 5 M; Alltech, Deerfield, IL) at a flow rate of 1 mL/min. Purified water and HPLC grade ACN each containing 0.1% formic acid were used as mobile phase A and B, respectively, with a linear gradient from 5% to 40% B over 50 min, followed by a ramp to 95% B in 10 min [30]. Fractions were manually collected every 1 min for 60 min.…”