Analysis of serum protein glycosylation by a differential lectin immunosorbant assay (dLISA)

Danni Li,Hanching Chiu,Daniel W Chan,Hui Zhang

doi:10.1186/1559-0275-10-12

Abstract

BackgroundLectin immunosorbant assays (LISAs) have been widely used for analyzing protein glycosylation. However, the analysis of serum samples by LISAs could suffer from high sample-dependent background noise. The aim of this study is to develop a differential lectin immunosorbant assay (dLISA) with reduced background interferences.MethodsFor the analysis of protein glycosylation, dLISA establishes a dose–response curve for every serum sample. The sample is split into five aliquots. Four aliquots undergo differential removal of the glycoprotein of interest by immunoprecipitation. Then, all five aliquots are subject to two measurements: protein by immunoassay and protein glycans by LISA. A dose–response curve is established by plotting glycans signals on the y-axis and protein levels on the x-axis for all the aliquots. Slope of the curve, calculated by linear progression analysis and expressed as fluorescence per concentration of protein, is used for the measurement of protein glycosylation in the serum sample.Results/conclusionsTo demonstrate the feasibility of the dLISA approach, we used recombinant, fucosylated tissue inhibitor of metallopeptidase 1 (TIMP-1) as the target glycoprotein. Magnetic beads based TIMP1 immunoassay and TIMP-1 UEA LISA were developed for the measurement of TIMP1 protein and terminal α1, 2 fucosylated glycans on TIMP1, respectively. Serum samples supplemented with differentially fucosylated recombinant TIMP-1 were used to demonstrate that the slopes measured the TIMP-1 fucosylation, and were less prone to background interference.

Highlights

Aberrant glycosylation of proteins has been implicated in many human diseases [1,2,3]
Magnetic beads coupled with tissue inhibitor of metallopeptidase 1 (TIMP-1) antibodies are used for the removal of endogenous TIMP-1 in these aliquots: A1 is left untreated, whereas A2 to A5 are treated with increasing amounts of magnetic beads for the differential removal; the amount of beads used for A5 is sufficient for the complete removal of the endogenous TIMP-1
To tease out the portions of the signals that are specific for TIMP-1, the TIMP-1 Ulex europaeus agglutinin (UEA) Lectin immunosorbant assays (LISAs) signals of A1 to A5 are plotted on the y-axis against their respective TIMP-1 protein levels on the x-axis to establish a dose–response curve

Summary

Introduction

Aberrant glycosylation of proteins has been implicated in many human diseases [1,2,3]. Analytical tools combining MS with separation and enrichment techniques such as hydrophilic interaction chromatography (HILIC) and immunoaffinity enrichment are expected to provide a wealth of information on glycosylation changes that would allow a better understanding of the biological attributes of glycoproteins [10,11,12,13]. Complementary to the MS based techniques are affinitybased techniques for the analysis of glycosylation changes, such as lectin immunosorbant assays (LISAs) [4,6,14,15,16]. Due to lectins’ broad specificities, LISAs could provide additional information on all the glycans that the lectins bind to and resulting in better sensitivity for the detection of glycosylation changes of proteins. Lectin immunosorbant assays (LISAs) have been widely used for analyzing protein glycosylation. The aim of this study is to develop a differential lectin immunosorbant assay (dLISA) with reduced background interferences

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Methods

Results

Discussion

Conclusion