Optimized Workflow for Preparation of APTS-Labeled N-Glycans Allowing High-Throughput Analysis of Human Plasma Glycomes using 48-Channel Multiplexed CGE-LIF

Abstract

High-throughput methods for oligosaccharide analysis are required when searching for glycan-based biomarkers. Next to mass spectrometry-based methods, which allow fast and reproducible analysis of such compounds, further separation-based techniques are needed, which allow for quantitative analysis. Here, an optimized sample preparation method for N-glycan-profiling by multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) was developed, enabling high-throughput glycosylation analysis. First, glycans are released enzymatically from denatured plasma glycoproteins. Second, glycans are labeled with APTS using 2-picoline borane as a nontoxic and efficient reducing agent. Reaction conditions are optimized for a high labeling efficiency, short handling times, and only limited loss of sialic acids. Third, samples are subjected to hydrophilic interaction chromatography (HILIC) purification at the 96-well plate format. Subsequently, purified APTS-labeled N-glycans are analyzed by CGE-LIF using a 48-capillary DNA sequencer. The method was found to be robust and suitable for high-throughput glycan analysis. Even though the method comprises two overnight incubations, 96 samples can be analyzed with an overall labor allocation time of 2.5 h. The method was applied to serum samples from a pregnant woman, which were sampled during first, second, and third trimesters of pregnancy, as well as 6 weeks, 3 months, and 6 months postpartum. Alterations in the glycosylation patterns were observed with gestation and time after delivery.

Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles—Part 1: Separation-based methods

“…28,[39][40][41][42] All other methods applied fluorescent labeling. Four methods used electrophoretic separation, which included conventional high-resolution capillary gel electrophoresis with laser-induced fluorescence [CE-LIF(APTS-HR1)], [43][44][45][46][47][48] DNA-sequencer-aided fluorophore-assisted carbohydrate electrophoresis after 8-aminopyrene-1,3,6-trisulfonic acid (APTS) labeling for high-throughput screening [DSA-FACE(APTS)], [49][50][51][52][53][54] high-resolution capillary gel electrophoresis with rapid labeling with APTS via reductive amination [CE-LIF (APTS-HR2)], and cartridge-based capillary gel electrophoresis with rapid 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) labeling, in development specifically for screening [CCGE(ANTS)]. The CE methods differ with regard to the labeling method: APTS labeling for the "normal" method requires 4 to 24 h for labeling, whereas rapid reductive amination has been optimized Three laboratories were involved in performing the experiments, an analytical laboratory in a development department, a quality control laboratory and a laboratory of a vendor of glycoanalytical tools.…”

Evaporative fluorophore labeling of carbohydrates via reductive amination

“…The reaction speed and yield of this acidcatalyzed labeling reaction is greatly influenced by the amount and type of acid used. Organic acids with low pK a , such as acetic acid (pK a = 4.75), malonic acid (pK a1 = 2.83) or citric acid (pK a1 = 3.15), are more commonly used to accelerate glycan labeling [27,28]. Studies have shown that stronger acids were able to increase derivatization yield [29], but their use was associated with higher sialic acid loss [30].…”

Recent Developments in Liquid Chromatography and Capillary Electrophoresis for the Analysis of Glycoprotein Glycans

“…Callewaert et al reported the application of a DNA sequencer to profiling N-glycans in serum glycoproteins; all sample preparation steps, including PNGase F digestion and labeling with APTS, were performed on a 96-well plate simultaneously, and the analysis of all samples was completed in half an hour. 151,152 …”