A relative and absolute quantification of neutral N-linked oligosaccharides using modification with carboxymethyl trimethylammonium hydrazide and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
Quantification of oligosaccharides is of great importance to investigate variations or changes in the glycans of glycoconjugates. Mass spectrometry (MS) has been widely applied to identification and structural analysis of complex oligosaccharides. However, quantification using MS alone is still quite challenging due to heterogeneous charge states and different ionization efficiency of various types of oligosaccharides. To overcome such shortcomings, derivatization with carboxymethyl trimethylammonium hydrazide (Girard's reagent T [GT]) was introduced to generate a permanent cationic charge at the reducing end of neutral oligosaccharides, resulting in mainly [M](+) ion using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), so that the ambiguities caused by metal adduct peaks such as [M+K](+) and [M+Na](+) were avoided. To verify our method, the relative and absolute quantification of neutral glycans from human immunoglobulin G (IgG) and ovalbumin with internal standards of dextran ladders using MALDI-TOF MS were compared with those performed by conventional normal-phase high-performance liquid chromatography (NP-HPLC) profiling. The quantification using GT derivatization and MALDI-TOF MS agreed well with the HPLC profiling data and showed excellent reliability and reproducibility with better resolution and sensitivity. This method was further applied to quantify the enzymatically desialylated N-glycans from miniature pig kidney membrane proteins. The results showed that the low-abundance structures that could not be resolved by NP-HPLC were quantified with high sensitivity. Thus, this novel method of using modification of neutral sugars with GT is quite powerful for neutral glycan analysis, especially to quantify minute glycan samples with undetectable levels using HPLC.
Gil
Geun-Cheol
GC
School of Chemical and Biological Engineering in College of Engineering, Seoul National University, Shillim-dong, Seoul 151-742, Korea.
Kim
Yun-Gon
YG
Kim
Byung-Gee
BG
D000818:Animals; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D006023:Glycoproteins / Q000737:chemistry; D006801:Humans; D006834:Hydrazines / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry; D007668:Kidney / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry; D015394:Molecular Structure; D009844:Oligosaccharides / Q000032:analysis* / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013552:Swine; D013556:Swine, Miniature; D062367:ortho-Aminobenzoates / Q000737:chemistry
D006023:Glycoproteins; D006834:Hydrazines; D007074:Immunoglobulin G; D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D011134:Polysaccharides; D062367:ortho-Aminobenzoates; C031385:anthranilic acid