Profile of native N-linked glycan structures from human serum using high performance liquid chromatography on a microfluidic chip and time-of-flight mass spectrometry

Abstract

Protein glycosylation involves the addition of monosaccharides in a stepwise process requiring no glycan template. Therefore, identifying the numerous glycoforms, including isomers, can help elucidate the biological function(s) of particular glycans. A method to assess the diversity of the N-linked oligosaccharides released from human serum without derivatization has been developed using on-line nanoLC and high resolution TOF MS. The N-linked oligosaccharides were analyzed with MALDI FT-ICR MS and microchip LC MS (HPLC-Chip/TOF MS). Two microfluidic chips were employed, the glycan chip (40 nL enrichment column, 43 x 0.075 mm(2) i.d. analytical column) and the high capacity chip (160 nL enrichment column, 140 x 0.075 mm(2) i.d. analytical column), both with graphitized carbon as the stationary phase. Both chips offered good sensitivity and reproducibility in separating a heterogeneous mixture of neutral and anionic oligosaccharides between injections. Increasing the length and volume of the enrichment and the analytical columns improved resolution of the peaks. Complex type N-linked oligosaccharides were the most abundant oligosaccharides in human serum accounting for approximately 96% of the total glycans identified, while hybrid and high mannose type oligosaccharides comprise the remaining approximately 4%.

Figure 1

MALDI FT MS of a) the neutral oligosaccharides (10% acetonitrile fraction), b) the neutral and anionic oligosaccharides (20% acetonitrile fraction), and c) the anionic oligosaccharides (40% acetonitrile fraction) released from human serum.

Figure 2

Collision induced dissociation of a) m/z 1648 with composition 1 deoxyhexose (dHex), 4 hexoses (Hex), and 4 N-acetylhexosamines (HexNAc) and b) m/z 1810 with composition 1 deoxyhexose (dHex), 5 hexoses (Hex), and 4 N-acetylhexosamines (HexNAc).

Figure 3

Base peak chromatograms of a) the neutral oligosaccharides, b) the neutral and anionic oligosaccharides, and c) the anionic oligosaccharides released from human serum. Each fraction has been annotated with the proposed structure corresponding to the most abundant oligosaccharide at that retention time.

Figure 4

Extracted ion chromatograms of neutral N-linked oligosaccharides released from human serum a & d) m/z 813, [M+2H]²⁺ with a composition of 1 dHex, 4 Hex, 4 HexNAc, b & e) m/z 894, [M+2H]²⁺ with a composition of 1 dHex, 5 Hex, 4 HexNAc, and c & f) m/z 996, [M+2H]²⁺ with a composition of 1 dHex, 5 Hex, 5 HexNAc. The chromatograms on the left column were obtained using the glycan chip (a–c) and those on the right column were obtained using the high capacity chip (d–f).

Figure 5

Extracted ion chromatograms of anionic N-linked oligosaccharides released from human serum a & c) m/z 967, [M+2H]²⁺ with a composition of 1 NeuAc, 5 Hex, 4 HexNAc and b & d) m/z 1039, [M+2H]²⁺ with a composition of 1 NeuAc, 1 dHex, 5 Hex, 4 HexNAc. The chromatograms on the left column were obtained using the glycan chip (a & b) and those on the right column were obtained using the high capacity chip (c & d).

Figure 6

Base peak chromatograms of a) the neutral oligosaccharides from triplicate digestion experiments, b) the neutral oligosaccharides from a single experiment after repeated injections of three using the glycan chip, and c) the neutral and anionic oligosaccharides after repeated injections of three using the high capacity chip.

Figure 7

Total ion chromatograms of the combined fractions of the N-linked oligosaccharides released from a) commercially purchased human serum, b) donor 1 serum, and c) donor 2 serum obtained using the high capacity chip.

Figure 8

Pie charts illustrating the N-linked oligosaccharide types and the saccharide compositions for a) commercially purchased human serum, b) donor 1 serum, and c) donor 2 serum obtained using the high capacity chip.