A carbon nanoparticles‐based solid‐phase purification method facilitating sensitive MALDI–MS analysis of permethylated N‐glycans

Abstract

In recent decades, MALDI–MS has been extensively used for the analysis of glycans. However, native glycans usually have low ionization efficiency in MS, which hinders the direct analysis. Permethylation of glycans is a solution for this issue, but a significant amount of salt is introduced during this process, which can further suppress the MS signals. Thus, it is necessary to purify the glycans prior to MALDI–MS analysis. In this study, we developed a carbon nanoparticles‐based solid‐phase purification method to enable direct MALDI–MS analysis of permethylated glycans. Two carbon nanomaterials, carbon nanoparticles (CNPs) and graphene nanosheets (GNs), and two conventional carbon materials, activated charcoal and porous graphitic carbon (PGC), were investigated as sorbents to purify permethylated N‐glycans derived from ribonuclease B and fetuin. The results confirmed the superior performance of CNPs over the other carbon materials. Additionally, our method was also employed to purify glycans released from human sera in different esophageal disease stages. The obtained data confirmed 16 and 18 structures in adenocarcinoma and Barret's sera with significantly different relative intensities versus disease‐free sera. Comparing the performance of CNPs‐based solid‐phase purification method employed in this study to online purification suggested more than 97% recovery rate. The results of this study demonstrate that CNPs have the potential to be a better alternative to existing solid‐phase purification sorbents.

Comparative Membrane N-Glycomics of Different Breast Cancer Cell Lines To Understand Breast Cancer Brain Metastasis

“…The reduced N -glycans were then permethylated using the previously reported solid-phase permethylation approach. − Dried N -glycan samples were dissolved in 51.2 μL of loading solution (30 μL of DMSO, 1.2 μL of water, and 20 μL of iodomethane). The spin columns were loaded with sodium hydroxide beads (suspended in DMSO) and spun down at 300 g for 2 min.…”

Glycome Profiling of Cancer Cell Lines Cultivated in Physiological and Commercial Media

“…After dialysis, samples were dried and reduced by 10 µL of borane-ammonia complex solution (10 mg/mL) at 60 ℃ for 1 h [ 32 ]. Reduced glycans were subjected to solid-phase permethylation [ 33 , 34 , 35 , 36 , 37 ]. Briefly, the reduced and dried glycans were suspended in 30 µL DMSO, 1.2 µL water, and 20 µL iodomethane.…”

N-Linked Surface Glycan Biosynthesis, Composition, Inhibition, and Function in Cnidarian-Dinoflagellate Symbiosis

“…With the knowledge that N-glycan biosynthesis pathways are present and even conserved in Breviolum minutum, we sought to characterize the N-linked glycome. We harvested algal cells, cleaved the N-linked surface glycans, and after per-methylation used LC-MS/MS for glycan analysis [50][51][52][53]63]. Based on molecular weight, retention time, and comparison to carbohydrate standards, the overall cleaved N-linked glycan sample exhibited 52% high-mannose glycans, 12% core-fucosylated glycans, and, to our surprise, 3% sialylated glycans (Figure 2a).…”

“…For enzymatic treatment, 1 L of culture was spun down and treated with Peptide:N-glycosidase F for 72 h (50 U PNGase F • mL -1 f/2 media). The reactions were stirred at 37 C for 72 h. The glycan sample was dialyzed using 10k MWCO dialysis tubing in mL ultrapure water; water was changed three times over the course of h. The glycan sample was lyophilized and 1 mg of lyophilized sample was reduced and permethylated as previously reported [50][51][52][53]. In summary, lyophilized samples were dissolved in 10 µL of borane-ammonia complex solution (10 mg• mL -1 ) and incubated in the water bath at 60 o C for 1h.…”