Capillary Electrophoresis-Electrospray Ionization-Mass Spectrometry for Quantitative Analysis of Glycans Labeled with Multiplex Carbonyl-Reactive Tandem Mass Tags

Abstract

Recently developed carbonyl-reactive aminoxy tandem mass tag (aminoxyTMT) reagents enable multiplexed characterization and quantitative comparison of structurally complex glycans between different biological samples. Compared to some previously reported isotopic labeling strategies for glycans, the use of the aminoxyTMT method features a simple labeling procedure, excellent labeling efficiency, and reduced spectral complexity at the MS(1) level. Presence of the tertiary amine functionality in the reporter region of the aminoxyTMT labels leads to increased ionization efficiency of the labeled glycans thus improving electrospray ionization (ESI)-mass spectrometry (MS) detection sensitivity. The use of the labeling reagent also makes electrophoretic separation of the labeled neutral and acidic glycans feasible. In this work, we characterized the ESI and collision induced dissociation (CID) behavior of the aminoxyTMT-labeled neutral and sialylated glycans. For the high-mannose N-glycans and small sialylated oligosaccharides, CID fragmentation of [M + Na + H](2+) provides the most informative MS(2) spectra for both quantitative and qualitative analysis. For complex N-glycans, MS(3) of the protonated Y1(H) ion can be used for relative quantification without interference from the HexNAc fragments. Online capillary electrophoresis (CE)-ESI-MS/MS analyses of multiplexed aminoxyTMT-labeled human milk oligosaccharides (HMOs) and different types of N-glycans released from glycoprotein standards were demonstrated. Improved resolution and quantification accuracy of the labeled HMO isomers was achieved by coupling CE with traveling wave ion mobility (TWIM)-CID-MS/MS. N-Glycans released from human serum protein digests were labeled with six-plex aminoxyTMT and subjected to CE-ESI-MS/pseudo-MS(3) analysis, which demonstrated the potential utility of this glycan relative quantification platform for more complex biological samples.

Direct comparison of derivatization strategies for LC-MS/MS analysis ofN-glycans

“…Quantitative glycomics can also be achieved using multiplexing reagent derivatization. Multiplexing reagents, such as heavy/light 2-AB, aminoxyTMT, 18, 19 iARTs, 20 QUANTITY 21 and INLIGHT, 22 have been reported for quantitative glycomics based on full MS or MS/MS reporter ions. Multiplexed LC-MS strategies reduce analysis time by analyzing multiple samples at once and eliminate the variation induced by ionization efficiency fluctuation and MS response bias.…”

Microfluidic Capillary Electrophoresis–Mass Spectrometry for Analysis of Monosaccharides, Oligosaccharides, and Glycopeptides

“…While CE has been shown to resolve isomeric glycans, such large differences in migration times for the same glycan composition were not expected. 46 We hypothesized that the increased migration times and elution in multiple peaks was related to a combination of increased glycan net negative charge, increased hydrodynamic radius, and nonspecific interactions of acidic glycans with the positively charged capillary coating. To demonstrate the effect of number of sialic acids on migration times, overlaid extracted ion traces have been presented in Figure S-7; the EIEs have been color-coded to reflect the number of NeuAc in glycan compositions.…”

Recent advances in ion mobility–mass spectrometry for improved structural characterization of glycans and glycoconjugates

“…Because mobility separations typically occur on the order of milliseconds it is possible to couple IM between LC [36,37] or CE [38] and mass spectrometry for glycan analysis (Figure 2a). The combination of orthogonal separation methods has been demonstrated to offer improved characterization of isomeric glycans.…”