N-glycosylamine-mediated isotope labeling for mass spectrometry-based quantitative analysis of N-linked glycans.

N-linked glycosylation is a major protein modification involved in many essential cellular functions. Methods capable of quantitative glycan analysis are highly valuable and have been actively pursued. Here we describe a novel N-glycosylamine-based strategy for isotopic labeling of N-linked glycans for quantitative analysis by use of mass spectrometry (MS). This strategy relies on the primary amine group on the reducing end of freshly released N-linked glycans for labeling, and eliminates the need for the harsh labeling reaction conditions and/or tedious cleanup procedures required by existing methods. By using NHS-ester amine chemistry we used this strategy to label N-linked glycans from a monoclonal antibody with commercially available tandem mass tags (TMT). Only duplex experiments can be performed with currently available TMT reagents, because quantification is based on the intensity of intact labeled glycans. Under mild reaction conditions, greater than 95% derivatization was achieved in 30 min and the labeled glycans, when kept at -20 °C, were stable for more than 10 days. By performing glycan release, TMT labeling, and LC-MS analysis continuously in a single volatile aqueous buffer without cleanup steps, we were able to complete the entire analysis in less than 2 h. Quantification was highly accurate and the dynamic range was large. Compared with previously established methods, N-glycosylamine-mediated labeling has the advantages of experimental simplicity, efficient labeling, and preserving glycan integrity.

High-throughput multimodal strong anion exchange purification and N-glycan characterization of endogenous glycoprotein expressed in glycoengineered Pichia pastoris.

The secretory pathway of the yeast Pichia pastoris has been engineered to produce complex human-type N-glycans (Choi et al., Proc Natl Acad Sci USA 100:5022-5027, 2003; Hamilton et al., Science 301:1244-1246, 2003; Hamilton et al., Science 313:1441-1443, 2006). In contrast to the heterogeneous glycans produced on the therapeutic glycoproteins expressed in mammalian cell lines, glycoengineered P. pastoris can be designed to produce a specific, preselected glycoform. In order to achieve glycan uniformity on the target protein, No Open Reading Frame (NORF) yeast cell lines are screened extensively during various stages of glycoengineering. In the absence of the target protein of interest, screening the NORF yeast cell lines for glycoform uniformity becomes a challenge. The common approach so far has been to analyze the total cell glycan pool released from glycoproteins of the NORF yeast cells to predict the N-glycan uniformity. As this does not always accurately predict the N-glycan end product, we describe in this chapter a detailed protocol for a non-affinity-based high-throughput purification of an endogenous glycoprotein. This protein of interest has been introduced during the early stages of glycoengineering process and its N-glycan profile is utilized as a tool for glycoengineering screening.