The development of retrosynthetic glycan libraries to profile and classify the human serum N-linked glycome

Abstract

Annotation of the human serum N-linked glycome is a formidable challenge but is necessary for disease marker discovery. A new theoretical glycan library was constructed and proposed to provide all possible glycan compositions in serum. It was developed based on established glycobiology and retrosynthetic state-transition networks. We find that at least 331 compositions are possible in the serum N-linked glycome. By pairing the theoretical glycan mass library with a high mass accuracy and high-resolution MS, human serum glycans were effectively profiled. Correct isotopic envelope deconvolution to monoisotopic masses and the high mass accuracy instruments drastically reduced the amount of false composition assignments. The high throughput capacity enabled by this library permitted the rapid glycan profiling of large control populations. With the use of the library, a human serum glycan mass profile was developed from 46 healthy individuals. This paper presents a theoretical N-linked glycan mass library that was used for accurate high-throughput human serum glycan profiling. Rapid methods for evaluating a patient's glycome are instrumental for studying glycan-based markers.

Figure 1

Starting N-linked glycan structures prior to simulated retrosynthetic degradation. High mannose (left), hybrid (center), complex (right). The hexasaccharide N-linked core is boxed in the high mannose diagram.

Figure 2

(a). Complex retrosynthetic state-transition network prior to fucose addition. The chitobiose core was omitted in the figure for clarity. Representative bi-antennary, tri-antennary and tetra-antennary glycan degradation families are highlighted. Figure 2(b). High mannose retrosynthetic state-transition network prior to fucose addition. The chitobiose core was omitted in the figure for clarity. Figure 2(c). Hybrid retrosynthetic state-transition network prior to fucose addition. The chitobiose core was omitted in the figure for clarity.

Figure 2

(a). Complex retrosynthetic state-transition network prior to fucose addition. The chitobiose core was omitted in the figure for clarity. Representative bi-antennary, tri-antennary and tetra-antennary glycan degradation families are highlighted. Figure 2(b). High mannose retrosynthetic state-transition network prior to fucose addition. The chitobiose core was omitted in the figure for clarity. Figure 2(c). Hybrid retrosynthetic state-transition network prior to fucose addition. The chitobiose core was omitted in the figure for clarity.

Figure 2

(a). Complex retrosynthetic state-transition network prior to fucose addition. The chitobiose core was omitted in the figure for clarity. Representative bi-antennary, tri-antennary and tetra-antennary glycan degradation families are highlighted. Figure 2(b). High mannose retrosynthetic state-transition network prior to fucose addition. The chitobiose core was omitted in the figure for clarity. Figure 2(c). Hybrid retrosynthetic state-transition network prior to fucose addition. The chitobiose core was omitted in the figure for clarity.

Figure 3

Zoom of theoretical spectrum (top) real serum spectrum (bottom). The library was converted to Na+ adducts to compare with the MALDI spectrum of serum glycans.

Figure 4

A serum mass spectrum superimposed on top of the monoisotopic theoretical library (vertical bars). This figure demonstrates the importance of correct monoisotopic peak assignments prior to glycan assignments. There are three options when an experimental ion is in accord with the theoretical library: (1) The ion is a monoisotopic ion (Dots) and correctly annotated, (2) The ion is an isotope peak (Triangles) and is not annotated or (3) The ion is not detectable above the noise threshold (horizontal dotted line) and is not annotated.

Figure 5

Frequency of detection vs. N-linked glycan mass profile. The y-axis is the frequency of detection of an N-linked glycan mass out of 46 spectra while the x-axis is the glycan mass. The x-axis is sorted from the most frequently detected to the least.