Characterization of protein glycosylation using chip-based infusion nanoelectrospray linear ion trap tandem mass spectrometry

Abstract

Mass spectrometry (MS) has the potential to revolutionize structural glycobiology and help in the understanding of how post-translation events such as glycosylation affect protein activities. Several approaches to determine the structure of glycopeptides have been used successfully including fast atom bombardment, matrix-assisted laser desorption ionization, and electrospray ionization with a wide variety of mass analyzers. However, the identification of glycopeptides in a complex mixture still remains a challenge. The source of this challenge is primarily due to the poor ionization efficiency and rapid degradation of glycopeptides. In this report we describe the use of a chip-based infusion nanoelectrospray ionization technique in combination with a recently developed linear ion trap for identification and characterization of glycosylation in complex mixtures. Two standard synthetic glycans were analyzed using multiple-stage fragmentation analysis in both positive and negative ionization modes. In addition, the high mannose type N-glycosylation in ribonuclease B (RNase B) was used to map the glycosylation site and obtain the glycan structures. We were able to map the glycosylation site and obtain the glycan structures in RNase B in a single analysis. The results reported here demonstrate that the fully automated chip-based nanoelectrospray linear ion trap platform is a valuable system for oligosaccharide analyses due to the unique MS/MS and MS(n) capability of the linear ion trap and the extended analysis time provided by the ionization technique.

FIGURE 1A–E

Full-scan mass spectrum (A) of the biantennary N-linked core pentasaccharide at a concentration of 1 pmol/μL as described in Materials and Methods. B–E represent MS² through MS⁵ mass spectra derived by collision-induced fragmentation of (M + 2H)²⁺ at m/z 933.0 (B), m/z 933.0 → m/z 730.0 (C), m/z 933.0 → m/z 730.0 → m/z 527.0 (D), and m/z 933.0 → m/z 730.0 → m/z 527.0 → m/z 365.0 (E) for the structural analysis of the oligosaccharide. The arrows in red across the sugars (C–E) denote the glycosidic bonds where the cross-ring fragmentation occurred.

FIGURE 1A–E

Full-scan mass spectrum (A) of the biantennary N-linked core pentasaccharide at a concentration of 1 pmol/μL as described in Materials and Methods. B–E represent MS² through MS⁵ mass spectra derived by collision-induced fragmentation of (M + 2H)²⁺ at m/z 933.0 (B), m/z 933.0 → m/z 730.0 (C), m/z 933.0 → m/z 730.0 → m/z 527.0 (D), and m/z 933.0 → m/z 730.0 → m/z 527.0 → m/z 365.0 (E) for the structural analysis of the oligosaccharide. The arrows in red across the sugars (C–E) denote the glycosidic bonds where the cross-ring fragmentation occurred.

FIGURE 1A–E

Full-scan mass spectrum (A) of the biantennary N-linked core pentasaccharide at a concentration of 1 pmol/μL as described in Materials and Methods. B–E represent MS² through MS⁵ mass spectra derived by collision-induced fragmentation of (M + 2H)²⁺ at m/z 933.0 (B), m/z 933.0 → m/z 730.0 (C), m/z 933.0 → m/z 730.0 → m/z 527.0 (D), and m/z 933.0 → m/z 730.0 → m/z 527.0 → m/z 365.0 (E) for the structural analysis of the oligosaccharide. The arrows in red across the sugars (C–E) denote the glycosidic bonds where the cross-ring fragmentation occurred.

FIGURE 2A–C

Full-scan mass spectrum (A) of cellohexaose at a concentration of 1 pmol/μL as described in Materials and Methods. MS² and MS³ mass spectra derived by collision-induced fragmentation of (M – 2H)^2– at m/z 989.0 (B), m/z 989.0 → m/z 827.0 (C) for the structural analysis of the oligosaccharide.

FIGURE 2A–C

Full-scan mass spectrum (A) of cellohexaose at a concentration of 1 pmol/μL as described in Materials and Methods. MS² and MS³ mass spectra derived by collision-induced fragmentation of (M – 2H)^2– at m/z 989.0 (B), m/z 989.0 → m/z 827.0 (C) for the structural analysis of the oligosaccharide.

FIGURE 3A–F

Full-scan mass spectrum (A) of the tryptic digest of pancreatic ribonuclease B at a concentration of 1 pmol/μL as described in Materials and Methods. Tandem mass spectra derived by collision-induced dissociation of the (M + 2H)²⁺ precursor ion of the ribonuclease glycopeptides, m/z 1171.1 (B), m/z 1090.8 (C), m/z 1008.4 (D), m/z 927.5 (E), m/z 846.0 (F).

FIGURE 3A–F

Full-scan mass spectrum (A) of the tryptic digest of pancreatic ribonuclease B at a concentration of 1 pmol/μL as described in Materials and Methods. Tandem mass spectra derived by collision-induced dissociation of the (M + 2H)²⁺ precursor ion of the ribonuclease glycopeptides, m/z 1171.1 (B), m/z 1090.8 (C), m/z 1008.4 (D), m/z 927.5 (E), m/z 846.0 (F).

FIGURE 3A–F

Full-scan mass spectrum (A) of the tryptic digest of pancreatic ribonuclease B at a concentration of 1 pmol/μL as described in Materials and Methods. Tandem mass spectra derived by collision-induced dissociation of the (M + 2H)²⁺ precursor ion of the ribonuclease glycopeptides, m/z 1171.1 (B), m/z 1090.8 (C), m/z 1008.4 (D), m/z 927.5 (E), m/z 846.0 (F).

FIGURE 4A–C

MS³ through MS⁵ mass spectra derived by collision-induced fragmentation of (M + 2H)²⁺at m/z 846.0 → m/z 765.0 (A), m/z 846.0 → m/z 765.0 → m/z 684.0 (B), m/z 846.0 → m/z 765.0 → m/z 684.0 → m/z 603.0 (C) for the stepwise removal of terminal mannose residues.

FIGURE 4A–C

MS³ through MS⁵ mass spectra derived by collision-induced fragmentation of (M + 2H)²⁺at m/z 846.0 → m/z 765.0 (A), m/z 846.0 → m/z 765.0 → m/z 684.0 (B), m/z 846.0 → m/z 765.0 → m/z 684.0 → m/z 603.0 (C) for the stepwise removal of terminal mannose residues.

FIGURE 5

MS⁴ mass spectra derived by collision-induced fragmentation of (M + 2H)²⁺, m/z 846.0 → m/z 678.0 → m/z 475 for the identification of the amino acid sequence of the ribonuclease glycopeptide.