Carbon Nanoparticles and Graphene Nanosheets as MALDI Matrices in Glycomics: a New Approach to Improve Glycan Profiling in Biological Samples

Abstract

Glycomics continues to be a highly dynamic and interesting research area due to the need to comprehensively understand the biological attributes of glycosylation in many important biological functions such as the immune response, cell development, cell differentiation/adhesion, and host-pathogen interactions. Although matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) has proven to be suitable for glycomic profiling studies, there is a need for improved sensitivity in the detection of native glycans, which ionize inefficiently. In this study, we investigated the efficiencies of graphene nanosheets (GNs) and carbon nanoparticles (CNPs) as MALDI matrices and co-matrices in glycan profiling. Our results indicated an enhancement of signal intensity by several orders of magnitude upon using GNs and CNPs in MALDI analysis of N-glycans derived from a variety of biological samples. Interestingly, increasing the amounts of CNPs and GNs improved not only the signal intensities but also prompted in-source decay (ISD) fragmentations, which produced extensive glycosidic and cross-ring cleavages. Our results indicated that the extent of ISD fragmentation could be modulated by CNP and GN concentrations, to obtain MS² and pseudo-MS³ spectra. The results for glycan profiling in high salt solutions confirmed high salt-tolerance capacities for both CNPs and GNs. Finally, the results showed that by using CNPs and GNs as co-matrices, DHB crystal formation was more homogeneous which improved shot-to-shot reproducibility and sensitivity. Graphical Abstract ᅟ.

Keywords: Glycans; Graphene; MALDI-MS; Nanoparticles.

Figure 1.

The optical images of the spots (a) DHB dried at atmospheric pressure (b) DHB dried under vacuum (c) CNPs dried at atmospheric pressure (d) DHB+CNPs, dried at atmospheric pressure (e) GNs dried at atmospheric pressure (f) DHB+GNs, dried at atmospheric pressure.

Figure 2.

MALDI-TOF-MS profiles of N-glycans derived from 100 ng of RNase B using (a) 10μg DHB (b) 1μg CNPs (c) 1μg GNs (d) 10μg DHB+0.025μg CNPs (e) 10μg DHB+0.05μg GNs. Symbols: , N-acetylglucosamine; , Galactose; , Fucose; , Mannose; , N-acetylneuraminic acid.

Figure 3.

MALDI-TOF-MS profiles of RNase B glycans using DHB mixed with different amounts of (a) CNPs (b) GNs. Relative abundance of RNase B glycans recorded using DHB mixed with different amounts of (c) CNPs and (d) GNs.

Figure 4.

(a) MALDI-TOF-MS profile of N-glycans derived from 1μg of RNase B using 10μg DHB+ 0.5μg CNPs. Underlines indicate the five intact high mannose structures and the arrows designate the fragmented ^0,2A₅ ions corresponding to the five high mannose structures. (b) MS/MS spectrum of the ISD fragmented ion at m/z=1096.3 and (c) MS/MS spectrum of the ISD fragmented ion at m/z=1318.4. Symbols as in Figure 2.

Figure 5.

MALDI-TOF-MS profiles of (a) N-glycans derived from 500 ng of fetuin using 10μg DHB (b) N-glycans derived from 500 ng of fetuin using 10μg DHB+ 0.025 CNPs (c) permethylated N-glycans derived from 100 ng of RNase B, using 10μg DHB (d) permethylated N-glycans derived from 100 ng of RNase B, using 10μg DHB+ 0.025 CNPs (e) permethylated N-glycans derived from 100 ng of fetuin using 10μg DHB (f) permethylated N-glycans derived from 100 ng of fetuin, using 10μg DHB+ 0.025 CNPs. Symbols as in Figure 2.

Figure 6.

MALDI-TOF-MS profiles of purified and extracted permethylated glycans using CNPs, derived from (a) 0.8 μg mixture of RNase B, fetuin, and AGP with a 1:2:5 weigh ratio, respectively, (b) 10μl of human blood serum. Symbols as in Figure 2.