Investigation of different combinations of derivatization, separation methods and electrospray ionization mass spectrometry for standard oligosaccharides and glycans from ovalbumin

Abstract

Derivatization procedures using 1-phenyl-3-methyl-5-pyrazolone (PMP) and 2-aminonaphthalene trisulfone (ANTS) were selected among a number of well known methods for labelling carbohydrates. PMP derivatives were selected owing to our laboratory's previous high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) experience with these, whereas the ANTS-labelled compounds were prepared for fluorophore-assisted carbohydrate electrophoresis (FACE) separation. ANTS-oligosaccharide standards were characterized to study their ionization patterns. Reversed-phase and normal-phase HPLC systems were coupled on-line with ESI-MS. Each necessitated its own mobile phase system which, in turn, imposed some important changes in the ionization conditions used and/or on the ionization patterns and spectra obtained. Following characterization of the intact glycoprotein ovalbumin with ESI-MS, its glycans were detached using the enzyme PNGase-F. The glycans were subjected to PMP and ANTS derivatization. It was very difficult to separate ANTS derivatives by reversed-phase HPLC owing to lack of retention, and normal-phase HPLC offered reasonable retention with limited separation. PMP compounds overall yielded better normal- and reversed-phase separations and improved sensitivity over the ANTS-labelled sugars, for which negative mode ESI had to be used. The combination of ESI of intact ovalbumin and ESI of PMP-glycans gave rise to the detection of over 20 different glycoforms, excluding the possible presence of structural isomers for each sugar composition detected.

Protein glycosylation analysis by liquid chromatography–mass spectrometry

“…1-Phenyl-3-methyl-5-pyrazolone (PMP)-labelling of N-glycans followed by RP-LC (4.6 mm i.d. ; running solvents containing 0.05% trifluoroacetic acid, elution with organic solvents) and ESI-MS with in-source decay fragment-ion analysis was performed by the group of Perreault [25,26]. Recently, an alternative derivatisation of glycans with phenylhydrazine in order to allow RP-LC-MS was introduced by the same group [27][28][29].…”

Oligosaccharide Analysis in Urine by MALDI-TOF Mass Spectrometry for the Diagnosis of Lysosomal Storage Diseases

“…Because oligosaccharides are difficult to ionize, a majority of the singly charged oligosaccharides with high molecular weight are missed by LC-MS. A MALDI-TOF method has also been described to analyze FOS after derivatization with 2-aminonaphthalene trisulfone (ANTS) (5 ). However, ANTS derivatization has limited analytical sensitivity because of the competition between the acidic character of the sulfone groups of ANTS and the basic properties of N-acetylglucosamine (GlcNAc) residues for ionization (6 ). In a recent multiinstitutional study comparing methods for profiling glycoprotein glycans, MALDItime-of-flight/time-of-flight (MALDI-TOF/TOF) of permethylated oligosaccharides yielded adequate quantification and provided good correlation between different glycomics laboratories, which may be related to the fact that permethylation protects the weak terminal glycosidic linkage (e.g., sialic acid) (7 ).…”

Detection and Quantitation of Afucosylated N-Linked Oligosaccharides in Recombinant Monoclonal Antibodies Using Enzymatic Digestion and LC-MS

“…Released oligosaccharides can be analyzed by high performance anion-exchange chromatography with pulsedamperometric detection (HPAEC-PAD) [8], matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) [9][10][11], and a graphitized carbon column with mass spectrometry [12,13]. A variety of chemistry has been employed to derivatise released oligosaccharides either to introduce a chromophore or flurophore for detection by absorption or fluorescence [14][15][16][17][18][19][20][21][22][23], or to introduce a positive charged group to enhance mass spectrometry response [20,[24][25][26]. Fluorescence labeling using 2-aminobenzamide (2-AB) or less frequently 2-aminobenzoic acid (2-AA) followed by normal-phase high performance liquid chromatography (NP-HPLC) separation has become the most commonly used method because of the high sensitivity, high resolution, and good reproducibility [21,[27][28][29].…”

“…Fluorescence labeling using 2-aminobenzamide (2-AB) or less frequently 2-aminobenzoic acid (2-AA) followed by normal-phase high performance liquid chromatography (NP-HPLC) separation has become the most commonly used method because of the high sensitivity, high resolution, and good reproducibility [21,[27][28][29]. In addition, many of the derivatization reagents add hydrophobicity to the otherwise hydrophilic oligosaccharides and allow them to be analyzed by reversed-phase HPLC (RP-HPLC) [14][15][16][17][18].…”