Normal-Phase Nanoscale Liquid Chromatography−Mass Spectrometry of Underivatized Oligosaccharides at Low-Femtomole Sensitivity

Abstract

We here describe the online liquid chromatography (LC) electrospray ionization mass spectrometry (MS) of underivatized glycans using a nanoscale normal-phase amide column at a flow rate of 300 nL/min. Retention on the amide column is based on polar interactions of the oligosaccharide hydroxyl groups with the stationary phase, and thus, the retention time predictably increases with elongation of the oligosaccharide chain. The system is characterized by its high chromatographic resolution, which routinely allows the separation of isobaric structures. Separation of oligosaccharide mixtures over a 1-h range permits the detailed characterization of the different species by multiple ion selection and fragmentation steps using ion trap MS. The here presented miniaturization of the online-LC system to the nanoscale in combination with ion trap MS allows the detection of oligosaccharide species in a mixture at low-femtomole sensitivity. Online normal-phase nano-LC-MS of complex oligosaccharide mixtures further facilitates the sensitive and detailed structural analysis of oligosaccharides by overcoming the need for cumbersome and time-consuming derivatization procedures such as reductive amination for labeling with hydrophobic fluorophores or labeling with tritium. The method should be useful for the sensitive and quick analysis of glycosylation patterns and individual oligosaccharides from biotechnologically produced glycoproteins as well as scarcely available biological samples.

Using a nanoelectrospray-differential mobility spectrometer-mass spectrometer system for the analysis of oligosaccharides with solvent selected control over ESI aggregate ion formation

“…Although ESI-MS is a popular choice for glycan analysis, poor ionization efficiency of many native glycan molecules in positive mode ESI has led researchers to investigate ways to improve glycan ionization [13][14][15][16]. Due to the poor basicity of glycans, formation of sufficient MH ϩ ion abundance in positive mode ESI is difficult to achieve [13][14][15][16]. Negative mode ESI offers ionization advantages for the analysis of glycans, however, positive mode ESI is also desirable due to its capability to generate different MS/MS glycan ion fragmentation than typically observed in negative mode ESI-MS/MS [17,18].…”

“…While this approach has been successful, it increases the complexity of sample preparation, such that many researchers have explored other means by which analysis of underivatized glycans can be performed in positive mode ESI [13,17,18,21]. It has been found that metal and ammonium ion complexes, such as MNa ϩ and MNH 4 ϩ , form readily with contaminant/residual cations in the ESI solution [14,20]. As a result, many of these glycan-cation complexes have been utilized as the molecular ions for analysis via the introduction of salts at low mM concentrations into the ESI solution to maintain consistent ionization efficiency and generate structurally informative MS/MS fragmentation patterns [13, 15- [15,18].…”

New hyphenated methodologies in high‐sensitivity glycoprotein analysis

“…However, this approach has been mainly based on the use of analytical columns employing very high flow rates, which adversely affect sensitivity. Recently, this problem has been rectified through the use of capillary amide columns at a flow rate of 300 nL/min, interfaced to IT-MS [74]. The use of nanoLC format permitted the detection of oligosaccharide mixtures at low-femtomole sensitivity.…”

“…The use of nanoLC format permitted the detection of oligosaccharide mixtures at low-femtomole sensitivity. The approach was validated using N-glycans derived from keyhole limpet hemocyanine and horseradish peroxidase [74].…”

Analysis of Saccharides by the Addition of Amino Acids

“…With an increase in the biological importance of oligo-or polysaccharides associated with cell-cell recognition, protein targeting, and metabolic diseases, polysaccharide and glycol-conjugate research has been attracting more attention [4,5]. Although different ionization methods [6][7][8][9][10][11][12][13] have been developed for the analysis of saccharides, matrix-assisted laser desorption/ionization (MALDI) [8] and ESI [11,[14][15][16][17][18] are the most used ionization methods. MALDI time-of-flight (TOF) MS has been used for the analysis of saccharides, and the sensitivity has been improved to the low femtomole level by reductive amination of the samples [19][20][21][22][23].…”