Automated Preparation of MS-Sensitive Fluorescently Labeled N-Glycans with a Commercial Pipetting Robot

N-Glycan analysis is routinely performed for biotherapeutic protein characterization. A recently introduced N-glycan analysis kit using RapiFluor-MS (RFMS) labeling provides time savings over reductive amination labeling methods while also providing enhanced fluorescence (FLR) and mass spectrometry (MS) responses. This article demonstrates the semiautomation of this kit using an Andrew Alliance pipetting robot that promises further gains in productivity. This robotic platform uses standard manual pipettors and an optically guided arm to facilitate the automation of manual procedures. The manual RFMS protocol includes two heating and cooling steps during protein denaturation and de-N-glycosylation. However, the current Andrew Alliance automated platform cannot move reaction tubes to and from different heating blocks. As a result, samples prepared using the automated procedure remain in a computer-controlled Peltier effect heating block, requiring reoptimization of denaturation and de-N-glycosylation temperatures. Using hydrophilic interaction liquid chromatography to monitor the RFMS-labeled glycan profiles, the authors demonstrated the reproducibility of the automated protocol with percent relative standard deviations (RSDs) of 9%-19% for the total area and 0.8%-20% for the relative areas of major and minor glycoforms. Overall, the automated platform presented here proves to be a convenient and reliable solution for N-glycan preparation and analysis.