Quantitative twoplex glycan analysis using12C6and13C6stable isotope 2-aminobenzoic acid labelling and capillary electrophoresis mass spectrometry.
Capillary electrophoresis (CE) offers excellent efficiency and orthogonality to liquid chromatographic (LC) separations for oligosaccharide structural analysis. Combination of CE with high resolution mass spectrometry (MS) for glycan analysis remains a challenging task due to the MS incompatibility of background electrolyte buffers and additives commonly used in offline CE separations. Here, a novel method is presented for the analysis of 2-aminobenzoic acid (2-AA) labelled glycans by capillary electrophoresis coupled to mass spectrometry (CE-MS). To ensure maximum resolution and excellent precision without the requirement for excessive analysis times, CE separation conditions including the concentration and pH of the background electrolyte, the effect of applied pressure on the capillary inlet and the capillary length were evaluated. Using readily available12/13C6stable isotopologues of 2-AA, the developed method can be applied for quantitative glycan profiling in a twoplex manner based on the generation of extracted ion electropherograms (EIE) for12C6'light' and13C6'heavy' 2-AA labelled glycan isotope clusters. The twoplex quantitative CE-MS glycan analysis platform is ideally suited for comparability assessment of biopharmaceuticals, such as monoclonal antibodies, for differential glycomic analysis of clinical material for potential biomarker discovery or for quantitative microheterogeneity analysis of different glycosylation sites within a glycoprotein. Additionally, due to the low injection volume requirements of CE, subsequent LC-MS analysis of the same sample can be performed facilitating the use of orthogonal separation techniques for structural elucidation or verification of quantitative performance.
Characterisation and Comparability Laboratory, NIBRT-The National Institute for Bioprocessing Research and Training, Foster Avenue, Mount Merrion, Blackrock, Co. Dublin, Ireland.
Csaba
C
Váradi
Characterisation and Comparability Laboratory, NIBRT-The National Institute for Bioprocessing Research and Training, Foster Avenue, Mount Merrion, Blackrock, Co. Dublin, Ireland.
Stefan
S
Mittermayr
Characterisation and Comparability Laboratory, NIBRT-The National Institute for Bioprocessing Research and Training, Foster Avenue, Mount Merrion, Blackrock, Co. Dublin, Ireland.
Silvia
S
Millán-Martín
Characterisation and Comparability Laboratory, NIBRT-The National Institute for Bioprocessing Research and Training, Foster Avenue, Mount Merrion, Blackrock, Co. Dublin, Ireland. jonathan.bones@nibrt.ie.
Jonathan
J
Bones
D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000302:isolation & purification; D002240:Carbohydrate Sequence; D002247:Carbon Isotopes; D019075:Electrophoresis, Capillary / Q000379:methods*; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification*; D062367:ortho-Aminobenzoates / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D002247:Carbon Isotopes; D006023:Glycoproteins; D009844:Oligosaccharides; D062367:ortho-Aminobenzoates; C031385:anthranilic acid
2-Aminobenzoic acid; Capillary electrophoresis; Glycan analysis; Mass spectrometry; Quantitation; Stable isotope labelling