ABSTRACT
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.
Subject(s)
Automation, Laboratory/methods , Mass Spectrometry/methods , Polysaccharides/analysis , Proteins/chemistry , Robotics/instrumentation , Staining and Labeling/methods , Fluorescent Dyes/metabolism , Mass Spectrometry/instrumentation , Reproducibility of Results , Staining and Labeling/instrumentationABSTRACT
N-glycosylation of proteins is now routinely characterized and monitored because of its significance to the detection of disease states and the manufacturing of biopharmaceuticals. At the same time, hydrophilic interaction chromatography (HILIC) has emerged as a powerful technology for N-glycan profiling. Sample preparation techniques for N-glycan HILIC analyses have however tended to be laborious or require compromises in sensitivity. To address these shortcomings, we have developed an N-glycan labeling reagent that provides enhanced fluorescence response and MS sensitivity for glycan detection and have also simplified the process of preparing a sample for analysis. The developed labeling reagent rapidly reacts with glycosylamines upon their release from glycoproteins. Within a 5 min reaction, enzymatically released N-glycans are labeled with this reagent comprised of an NHS-carbamate reactive group, a quinoline fluorophore, and a tertiary amine for enhancing ESI+ MS ionization. To further expedite the released N-glycan sample preparation, rapid tagging has been integrated with a fast PNGase F deglycosylation procedure that achieves complete deglycosylation of a diverse set of glycoproteins in approximately 10 min. Moreover, a technique for HILIC-SPE of the labeled glycans has been developed to provide quantitative recovery and facilitate immediate HILIC analysis of the prepared samples. The described approach makes it possible to quickly prepare N-glycan samples and to incorporate the use of a fluorescence and MS sensitivity enhancing labeling reagent. In demonstration of these new capabilities, we have combined the developed sample preparation techniques with UHPLC HILIC chromatography and high sensitivity mass spectrometry to thoroughly detail the N-glycan profile of a monoclonal antibody.
Subject(s)
Analytic Sample Preparation Methods/methods , Fluorescent Dyes/chemistry , Hydrophobic and Hydrophilic Interactions , Polysaccharides/analysis , Polysaccharides/chemistry , Animals , Antibodies, Monoclonal/chemistry , Chromatography, Liquid , Glycoproteins/chemistry , Glycosylation , Humans , Immunoglobulin G , Indicators and Reagents/chemistry , Mice , Models, Molecular , Protein Conformation , Spectrometry, Fluorescence , Spectrometry, Mass, Electrospray Ionization , Time FactorsABSTRACT
Ionic analytes, such as peptides, can be challenging to separate by reverse-phase chromatography with optimal efficiency. They tend, for instance, to exhibit poor peak shapes, particularly when eluted with mobile phases preferred for electrospray ionization mass spectrometry. We demonstrate that a novel charged-surface C18 stationary phase alleviates some of the challenges associated with reverse-phase peptide separations. This column chemistry, known as CSH (charged-surface hybrid) C18, improves upon an already robust organosilica hybrid stationary phase, BEH (ethylene-bridged hybrid) C18. Based on separations of a nine-peptide standard, CSH C18 was found to exhibit improved loadability, greater peak capacities, and unique selectivity compared to BEH C18. Its performance was also seen to be significantly less dependent on TFA-ion pairing, making it ideal for MS applications where high sensitivity is desired. These performance advantages were evaluated through application to peptide mapping, wherein CSH C18 was found to aid the development of a high-resolution, high-sensitivity LC-UV-MS peptide mapping method for the therapeutic antibody, trastuzumab. From these results, the use of a C18 stationary phase with a charged surface, such as CSH C18, holds significant promise for facilitating challenging peptide analyses.
