Characterizing sialic acid variants at the glycopeptide level

Abstract

Beam-type collision-induced dissociation (CID) data of intact glycopeptides isolated from mouse liver tissue are presented to illustrate characteristic fragmentation of differentially sialylated glycopeptides. Eight glycoforms of an O-linked glycopeptide from Nucleobindin-1 are distinguished on the basis of the precursor masses and characteristic oxonium ions. We report that all sialic acid variants are prone to neutral loss from the charge reduced species in electron-transfer dissociation (ETD) fragmentation. We show how changes in sialic acid composition affect reverse phase chromatographic retention times: sialic acid addition increases glycopeptide retention times significantly; replacing the N-acetylneuraminic acid with the N-glycolyl variant leads to slightly reduced retention times, while O-acetylated sialic acid-containing glycoforms are retained longer. We then demonstrate how MS-Filter in Protein Prospector can use these diagnostic oxonium ions to find glycopeptides, by showing that a wealth of different glycopeptides can be found in a published phosphopeptide data set.

Figure 1

Low mass region of the beam-type CID (HCD) spectra of eight different glycoforms of a Nucleobindin-1 O-linked glycopeptide, ³¹AAPPQEDSQAgTETPDTGLYYHR⁵². Thr-41 indicated with a 'g' in the sequence was identified as the glycosylation site from ETD data. The structures in the middle of the panels indicate the glycan. The sugar-unit symbols are: GalNAc = yellow square; Gal = yellow circle; NeuAc = purple diamond; NeuGc = light blue diamond; Ac indicates O-acetylation of the sialic acid. The identity of the Hex modifying the GalNAc cannot be determined from the CID data: it was assumed that the most common, mucin-type core 1 structure, Gal-GalNAc modified this protein. The different sialic acids produced at least 2 diagnostic ions: a B1 fragment and a more abundant fragment formed via water loss from the B1 oxonium ion [Nomenclature: 29], as indicated in the appropriate panels. Diagnostic fragments that show the sialic acid attached to the core GalNAc are also labeled. For simplicity, fragments are only labeled in the panel where they are first observed. Panel 7 shows HCD data from a mixture spectrum: the tetrasaccharide featuring both sialic acid variants was detected in two differently acetylated forms.

Figure 2

ETD spectrum of ³¹AAPPQEDSQAgTETPDTGLYYHR⁵² peptide from Nucleobindin-1 bearing a NeuAcGal(NeuGc)GalNAc modification on Thr-41, i.e. glycoform 5 in Table 1 and Figure 1. ❖ labels the original precursor, m/z 1137.4800(3+), and its charge-reduced form. The three most intense fragment ions were formed via neutral losses of the different individual sialic acid residues and both sialic acids from the charge-reduced species. NeuAc = purple diamond; NeuGc = light blue diamond. Asterisks indicate z+1 fragments.

Figure 3

Superimposed extracted ion chromatograms of eight different glycoforms of ³¹AAPPQEDSQAgTETPDTGLYYHR⁵² from Nucleobindin-1. The glycosylation site has been assigned from ETD data and indicated with a 'g'. Monoisotopic masses listed in Table 1 were extracted with a mass accuracy of 20 ppm. The sugar-unit symbols are: GalNAc = yellow square; Gal = yellow circle; NeuAc = purple diamond; NeuGc = light blue diamond; Ac indicates O-acetylation of the sialic acid.

Figure 4

Superimposed extracted ion chromatograms of seven different glycoforms of the N-linked glycopeptide ¹⁷⁷VVLHPNHSVVDIGLIK¹⁹² from mouse Haptoglobin (see Table 3). The sugar-unit symbols are: GlcNAc = dark blue square; Man = green circle; Gal = yellow circle; Fuc = red triangle; NeuAc = purple diamond; NeuGc = light blue diamond; Ac indicates O-acetylation of the sialic acid. When it was not possible to determine at which branch the different sialic acids were attached they are shown 'disconnected'. Monoisotopic (4+) masses listed in Table 3 were extracted with a 20 ppm mass tolerance.

Figure 5

HCD spectrum of a glycopeptide acquired as part of a phosphopeptide analysis study [23]. The precursor ion was m/z 942.4100(4+). The presence of both NeuAc and NeuGc was ascertained from their diagnostic fragment ions (labeled). The glycan fragmentation allows determination that this is an O-linked glycopeptide featuring two isomeric glycan structures: NeuAcGal(NeuGc)GalNAc and NeuGcGal(NeuAc)GalNAc. The sialic acid positions within the trisaccharide fragments could not be determined, so are shown 'disconnected'. The identity of the peptide could not be determined from these data.