Chemo-Enzymatic Synthesis of (13)C Labeled Complex N-Glycans As Internal Standards for the Absolute Glycan Quantification by Mass Spectrometry.
Methods for the absolute quantification of glycans are needed in glycoproteomics, during development and production of biopharmaceuticals and for the clinical analysis of glycan disease markers. Here we present a strategy for the chemo-enzymatic synthesis of (13)C labeled N-glycan libraries and provide an example for their use as internal standards in the profiling and absolute quantification of mAb glycans by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. A synthetic biantennary glycan precursor was (13)C-labeled on all four amino sugar residues and enzymatically derivatized to produce a library of 15 glycan isotopologues with a mass increment of 8 Da over the natural products. Asymmetrically elongated glycans were accessible by performing enzymatic reactions on partially protected UV-absorbing intermediates, subsequent fractionation by preparative HPLC, and final hydrogenation. Using a preformulated mixture of eight internal standards, we quantified the glycans in a monoclonal therapeutic antibody with excellent precision and speed.
Glycotechnology Group, CIC biomaGUNE , Paseo Miramon 182, 20009, San Sebastian, Spain.
Begoña
B
Echeverria
Glycotechnology Group, CIC biomaGUNE , Paseo Miramon 182, 20009, San Sebastian, Spain.
Juan
J
Etxebarria
Glycotechnology Group, CIC biomaGUNE , Paseo Miramon 182, 20009, San Sebastian, Spain.
Nerea
N
Ruiz
Glycotechnology Group, CIC biomaGUNE , Paseo Miramon 182, 20009, San Sebastian, Spain.
Álvaro
Á
Hernandez
Mass Spectrometry Platform, CIC biomaGUNE , Paseo Miramon 182, 20009, San Sebastian, Spain.
Javier
J
Calvo
Pharma Biotech Development Penzberg, Roche Diagnostics GmbH , 82377 Penzberg, Germany.
Markus
M
Haberger
Pharma Biotech Development Penzberg, Roche Diagnostics GmbH , 82377 Penzberg, Germany.
Dietmar
D
Reusch
Glycotechnology Group, CIC biomaGUNE , Paseo Miramon 182, 20009, San Sebastian, Spain.|CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN) , Paseo Miramon 182, 20009 San Sebastian, Spain.
Niels-Christian
NC
Reichardt
D002247:Carbon Isotopes; D013058:Mass Spectrometry / Q000379:methods*; D011134:Polysaccharides / Q000032:analysis* / Q000096:biosynthesis* / Q000138:chemical synthesis / Q000737:chemistry; D012015:Reference Standards
D002247:Carbon Isotopes; D011134:Polysaccharides