Nanoscale reversed-phase liquid chromatography–mass spectrometry of permethylated N-glycans

Abstract

Reversed-phase liquid chromatography on the nanoscale coupled to electrospray tandem mass spectrometry was used to analyse a mixture of four commercial glycan standards, and the method was further adapted to N-glycans enzymatically released from alpha-1-acid glycoprotein and immunoglobulin gamma. Glycans were permethylated to enable their separation by reversed-phase chromatography and to facilitate interpretation of fragmentation data. Prior to derivatization of glycans by permethylation, they were reduced to cancel anomerism because, although feasible, it was not desired to separate α- and β-anomers. The effect of supplementing chromatographic solvent with sodium hydroxide to guide adduct formation was investigated. Raising the temperature in which the separation was performed improved chromatographic resolution and affected retention times as expected. It was shown by using the tetrasaccharides sialyl Lewis X and sialyl Lewis A that reversed-phase chromatography could achieve the separation of methylated isobaric glycan analytes. Isobaric glycans were detected among the N-glycans of immunoglobulin gamma and further analysed by tandem mass spectrometry.

Reversed-phase separation methods for glycan analysis

“…Dependent on whether electrospray ionization (ESI) MS is used or not, volatile buffers (sodium acetate, triethylammonium acetate, ammonium acetate, or ammonium formate) or nonvolatile buffers (sodium phosphate) are used. In two cases, low amounts of sodium hydroxide were added to solvent A to induce sodium adduct formation in MS [44, 46]. In addition, in a few methods an ion-pairing reagent (diethylamine and triethylamine) is used to support retention of glycans with charged groups such as sialic acids [60, 61].…”

“…In chromatography, small differences or double peaks could be observed for saccharides with a degree of polymerization of three monomers or more, because of these α and β anomers in the molecules. To overcome this without further labeling of the glycan, the reducing-end aldehyde in the open-ring conformation can be reduced with a reducing agent such as sodium borohydride, to form an alditol [29, 44, 120]. Other methods to eliminate double peaks (e.g., increasing the column temperature) exist but they will not be discussed in this review [10].…”

“…Glycans can also be derivatized by permethylation [43, 44, 48, 121, 122]. In contrast to the other derivatization methods discussed in this review, this derivatization does not occur at the reducing end.…”

Development of Monolithic Column Materials for the Separation and Analysis of Glycans

“…Labeling with 2-AP was first introduced by Hase in 1978 [170,171]. Permethylation of glycans can also be used to enable separation by RP-LC [172]. Labeling provides the hydrophobic interactions needed for retention and separation by the reversed phase column, and also a chromophore for enabling detection.…”

Small scale affinity purification and high sensitivity reversed phase nanoLC-MS N-glycan characterization of mAbs and fusion proteins

“…However, their approach resulted in long and time consuming gradients to achieve a good chromatographic resolution. Ritamo et al recently published on glycoanalysis utilizing nano-reversed phase chromatography (RPC) 21 . A nanoLC system was used to separate permethylated N-glycans, achieving separation for various structural isomers on a nano RP-column.…”