Enhanced detection of sulfated glycosylation sites in glycoproteins

Abstract

We demonstrate a method that enhances the mass spectral signal of mono-and disulfated glycopeptides, present in glycoproteins that contain many other nonsulfated glycoforms. This method utilizes the tripeptide Lys-Lys-Lys as an ion-pairing reagent to complex selectively to sulfated species, and enhance their ion signal. The method is applied to the analysis of glycopeptides released from the enzymatic digestion of ovine luteinizing hormone. In this analysis, a disulfated glycopeptide is identified that was previously not detectable by MS assays, and a monosulfated glycoform, present at less than 1% abundance, is identified without any separation or enrichment of these species prior to analysis. In addition to enhancing the ion signal of sulfated glycopeptides, the ion-pairing technique is useful in obtaining structural information about the sulfated species. (J Am Soc Mass Spectrom 2005, 16, 340 -348)

Distinguishing phosphorylation and sulfation in carbohydrates and glycoproteins using ion-pairing and mass spectrometry

“…Glycopeptides from ovine luteinizing hormone, ␣-subunit, (oLH␣) were produced in the laboratory of Dr. George Bousfield, Wichita State University, as described elsewhere [23,25].…”

“…In a previous study, the ion pairing approach was used to enhance the signal of mono-, di-sulfated glycopeptides from this protein [23]. Sulfated ion pairs can be readily observed in the ESI-MS analysis when adding the peptide K3 to the complex mixture [23].…”

“…In a previous study, the ion pairing approach was used to enhance the signal of mono-, di-sulfated glycopeptides from this protein [23]. Sulfated ion pairs can be readily observed in the ESI-MS analysis when adding the peptide K3 to the complex mixture [23]. This is because the peptide K3 suppresses the signal of neutral glycopeptides to facilitate the detection of sulfated glycoforms [23].…”

“…If the ion-pairing method, which distinguishes phosphorylated and sulfated monosaccharides, could be adapted to larger macromolecules, such as oligosaccharides and glycoproteins, this approach would afford a unique and effective way to distinguish phosphorylation from sulfation. Using ionpairing to discriminate these groups is advantageous to other methods because this approach provides significant advantages that are not related to phosphate/sulfate discrimination: The ion-pairing method has already been demonstrated to enhance the signal of sulfated glycopeptides [23], and to facilitate structural elucidation of the glycan moiety present in glycoproteins [24]. Thus, sulfate/ phosphate determination could be accomplished in a synergistic analysis that also enhances sensitivity and provides additional structural information about glycoproteins.…”

“…This is the first study to use the ion-pairing technique in conjunction with MS/MS to differentiate phosphorylation and sulfation on disaccharides and glycopeptides. In addition to discriminating the isobaric compounds, this approach can also simultaneously enhance the mass spectral signal of acidic groups in complex mixtures by enhancing the ionization efficiency of these functional groups [23].…”