Mesoporous Graphitized Carbon Column for Efficient Isomeric Separation of Permethylated Glycans

Abstract

Post-translational modifications are vital aspects of functional proteins. Therefore, it is critical to understand their roles in biological processes. Glycosylation is particularly challenging to study among these modifications due to the heterogeneity displayed by the glycans in terms of their isomers. Thus, researchers continue to strive for the development of efficient liquid chromatography techniques for isomeric separation of glycans. Porous graphitized carbon (PGC) nano column has been one of the most widely used columns for this purpose, but poor stability and lack of reproducibility led to its discontinuation. In our endeavor to find an alternative stationary phase for isomeric glycan separation, we tested the mesoporous graphitized carbon (MGC) material. Unprecedentedly, satisfactory results were obtained with a column only 1 cm long, which was tested on permethylated N-glycans derived from model glycoproteins as well as biological samples. The column was found to be reproducible across months as well as across different column preparations. Additionally, to decrease the dead volume and attain a better resolution, MGC was utilized to pack a 1 cm length of a pulled capillary nanospray emitter and again demonstrated efficient isomeric separation. Thus, MGC proved to be a suitable stationary phase to obtain efficient isomeric separation of permethylated N-glycans with 1 cm-long packing length, in both capillary columns and packed nanospray emitters.

Advances in mass spectrometry‐based glycomics—An update covering the period 2017–2021

“…Although the PGC column has been one of the most widely used columns for the isomeric separation of glycans, column fouling, instability, poor reproducibility, and needs for regenerations of this column were constant issues confronted by researchers [86,118,119]. These challenges eventually led to the cessation of the commercial nanoflow Hyper- carb PGC column.…”

“…In an attempt to find an alternative stationary phase to PGC, Mechref and coworkers [86] introduced an in‐house packed 1 cm long mesoporous graphitized carbon (MGC) column for the isomeric separation of glycans. The isomeric separation mechanism of glycans on MGC is assumed to be alike with the PGC columns; however, the smaller particle size (<500 nm) and pore size of the MGC column provide a more significant surface area for interaction with the analytes, resulting in better retention for glycans.…”

“…In addition, to decrease the dead volume and achieve a better resolution, MGC was also employed to pack a 1 cm length of a pulled capillary nanospray emitters by using the in‐house developed Peltier heater to control the temperature. Both MGC column and packed nanospray emitter are cost‐effective and favorable candidates for glycan isomeric separation studies [86].…”

“…However, due to the microheterogeneity and complexity of the glycan structures, isomeric separation of glycans is still lacking. Several LC-MS techniques have been employed to facilitate the isomeric separation of glycans including RP [80,81], HILIC [82,83], porous graphitic carbon (PGC) [84,85], and mesoporous graphitic carbon (MGC) [86]. This section discusses the most recent LC and CE methods that assist in the isomeric characterization and separation of glycans.…”

Isomeric separation of permethylated glycans by extra-long reversed-phase liquid chromatography (RPLC)-MS/MS

“…6,42,53,54 However, the relatively low reproducibility of PGC columns were observed. 55 In addition, the discontinue of this column by vendor hindered our further studies. Alternatively, we reported an in-house packed mesoporous graphitized carbon (MGC) column for isomeric separation of permethylated glycans.…”