Enhanced detection and identification of glycopeptides in negative ion mode mass spectrometry

Abstract

A combined mass spectrometry (MS) and tandem mass spectrometry (MS/MS) approach implemented with matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI FTICR MS) in the negative ion mode is described for enhanced glycopeptide detection and MS/MS analysis. Positive ion mode MS analysis is widely used for glycopeptide characterization, but the analyses are hampered by potential charge-induced fragmentation of the glycopeptides and poor detection of the glycopeptides harboring sialic acids. Furthermore, tandem MS analysis (MS/MS) via collision-induced dissociation (CID) of glycopeptides in the positive ion mode predominantly yields glycan fragmentation with minimal information to verify the connecting peptide moiety. In this study, glycoproteins such as, bovine lactoferrin (b-LF) for N-glycosylation and kappa casein (k-CN) for O-glycosylation were analyzed in both the positive- and negative ion modes after digestion with bead-immobilized Pronase. For the b-LF analysis, 44 potential N-linked glycopeptides were detected in the positive ion mode while 61 potential N-linked glycopeptides were detected in the negative ion mode. By the same token, more O-linked glycopeptides mainly harboring sialic acids from k-CN were detected in the negative ion mode. The enhanced glycopeptide detection allowed improved site-specific analysis of protein glycosylation and superior to positive ion mode detection. Overall, the negative ion mode approach is aimed toward enhanced N- and O-linked glycopeptide detection and to serve as a complementary tool to positive ion mode MS/MS analysis.

Figures

Figure 1

(a) Negative ion mode MALDI FT ICR MS spectrum of b-LF glycopeptides after 24 hrs immobilized pronase digestion. (b) Positive ion mode MS spectrum of the same b-LF glycopeptide sample. Inverted triangles () represent potential glycopeptide peaks labeled based on the GP finder analysis of the spectrum mass list, and circles () indicate dissociated glycan peaks.

Figure 2

(a) Negative ion mode MALDI FT ICR MS/MS spectrum of precursor ion with m/z 2034.70 Da assigned as [252NNS + GlcNAc2 + Man8 − H]−. (b) Positive ion mode MS/MS spectrum of corresponding sodiated peak with m/z 2058.68 Da assigned as [252NNS + GlcNAc2 + Man8 + Na]+. (c) Positive ion mode MS/MS spectrum of a di-sodiated peak with m/z 2080.68 Da assigned as [252NNS + GlcNAc2 + Man8 − H + 2Na]+. Circles () and squares () represent mannose and GlcNAc residues, respectively.

Figure 3

Negative ion mode MALDI FT ICR MS/MS spectrum of precursor ion with m/z 2196.75 Da assigned as [252NNS + GlcNAc2 + Man9 − H]−.

Figure 4

(a) Negative ion mode MALDI FT ICR MS spectrum of k-CN glycopeptides after 24 hrs of reaction with immobilized pronase digestion. (b) Positive ion mode MS spectrum of k-CN glycopeptides from the same sample. Inverted triangles () represent potential glycopeptide peaks labeled based on the GP finder analysis of the spectrum mass list. Rectangles (), circles () and diamond symbols () represent GalNAc, Gal and NeuAc residues, respectively.

Figure 5

(a) Negative ion mode MALDI FT ICR MS/MS spectrum of a k-CN glycopeptide with m/z 1517.61 Da assigned as [GEP152TS154TPT + GalNAc2 + Gal2 − H]−. (b) Positive ion mode MS/MS spectrum of the same glycopeptide with m/z 1541.61 Da and assigned as (GEP152TS154TPT + GalNAc2 + Gal2 + Na)+. Rectangles () and circles () represent GalNAc and Gal residues, respectively.

Figure 6

Glycosylation associated with each glycosite of bovine lactoferrin (b-LF). The various glycans at each site indicate glycan micro-heterogeneity in the occupancy of the five glycosites observed during the negative ion mode MS analysis of the b-LF glycopeptides. For each glycosite, the larger glycan structures represent corresponding glycopeptides with minimum relative intensities of 20% while the smaller glycan structures represent corresponding glycopeptides with relative intensities corresponding to less than 20%. Circles ( and ), squares () and triangles () represent hexose, GlcNAc and fucose residues, respectively.

Figure 7

Glycosylation associated with each glycosite of kappa casein (k-CN). The various glycans at each site indicate glycan micro-heterogeneity in the occupancy of the five glycosites observed during the negative ion mode MS analysis of the k-CN glycopeptides. Rectangles (), circles () and diamond symbols () represent GalNAc, Gal and NeuAc residues, respectively.