An HPLC-MALDI MS method for N-glycan analyses using smaller size samples: application to monitor glycan modulation by medium conditions.

Existing HPLC methods can provide detailed structure and isomeric information, but are often slow and require large initial sample sizes. In this study, a previously established two-dimensional HPLC technique was adapted to a two-step identification method for smaller sample sizes. After cleavage from proteins, purification, and fluorescent labeling, glycans were analyzed on a 2-mm reverse phase HPLC column on a conventional HPLC and spotted onto a MALDI-TOF MS plate using an automated plate spotter to determine molecular weights. A direct correlation was found for 25 neutral oligosaccharides between the 2-mm Shim-Pack VP-ODS HPLC column (Shimadzu) and the 6-mm CLC-ODS column (Shimadzu) of the standard two- and three-dimensional methods. The increased throughput adaptations allowed a 100-fold reduction in required amounts of starting protein. The entire process can be carried out in 2-3 days for a large number of samples as compared to 1-2 weeks per sample for previous two-dimensional HPLC methods. The modified method was verified by identifying N-glycan structures, including specifying two different galactosylated positional isomers, of an IgG antibody from human sera samples. Analysis of tissue plasminogen activator (t-PA) from CHO cell cultures under varying culture conditions illustrated how the method can identify changes in oligosaccharide structure in the presence of different media environments. Raising glutamine concentrations or adding ammonia directly to the culture led to decreased galactosylation, while substituting GlutaMAX-I, a dipeptide of L-alanine and L-glutamine, resulted in structures with more galactosylation. This modified system will enable glycoprofiling of smaller glycoprotein samples in a shorter time period and allow a more rapid evaluation of the effects of culture conditions on expressed protein glycosylation.