High‐throughput immunoaffinity enrichment and N‐glycan analysis of human plasma haptoglobin

Abstract

Haptoglobin (Hp) is a positive acute phase protein, synthesized in the liver, with four N-glycosylation sites carrying mainly complex type N-glycans. Its glycosylation is altered in different types of diseases but still has not been extensively studied mainly due to analytical challenges, especially the lack of a fast, efficient, and robust high-throughput Hp isolation procedure. Here, we describe the development of a high-throughput method for Hp enrichment from human plasma, based on monolithic chromatographic support in immunoaffinity mode and downstream Hp N-glycome analysis by hydrophilic interaction ultrahigh-performance liquid chromatography with fluorescent detection (HILIC-UHPLC-FLR). Chromatographic monolithic supports in a 96-well format enable fast, efficient, and robust Hp enrichment directly from diluted plasma samples. The N-glycome analysis demonstrated that a degree of Hp deglycosylation differs depending on the conditions used for N-glycan release and on the specific glycosylation site, with Asn 241 being the most resistant to deglycosylation under tested conditions. HILIC-UHPLC-FLR analysis enables robust quantification of 28 individual chromatographic peaks, in which N-glycan compositions were determined by UHPLC coupled to electrospray ionization quadrupole time of flight mass spectrometry. The developed analytical approach enables fast evaluation of total Hp N-glycosylation and is applicable in large-scale studies.

Probing Blood Plasma Protein Glycosylation with Infrared Spectroscopy

“…Fluorescent labeling was performed as previously described . Briefly, N -glycans were incubated at 65 °C for an hour with procainamide (43.2 mg/mL) as a fluorescent label and afterward an additional 1.5 h with 2-methylpiridine borane complex (44.8 mg/mL) as a reducing agent.…”

“…Fluorescent labeling was performed as previously described. 39 Briefly, N -glycans were incubated at 65 °C for an hour with procainamide (43.2 mg/mL) as a fluorescent label and afterward an additional 1.5 h with 2-methylpiridine borane complex (44.8 mg/mL) as a reducing agent. The fluorescent label and reducing agent were prepared in a 25 μL mixture of DMSO and glacial acetic acid (70:30, v/v) separately.…”

“…The wavelengths for excitation and emission were 310 and 370 nm, respectively. MS parameters were set as described previously 39 with the difference in mass range from 100 to 4000 m / z and a frequency of 0.5 Hz. N -Glycan structures in peaks were annotated in Bruker DataAnalysis 4.1. and determined from sum spectra created for 35 chromatographic peaks based on their retention time.…”