Comparison of Fucose-Specific Lectins to Improve Quantitative AFP-L3 Assay for Diagnosing Hepatocellular Carcinoma Using Mass Spectrometry

Abstract

Glycoproteins have many important biological functions. In particular, aberrant glycosylation has been observed in various cancers, such as liver cancer. A well-known glycoprotein biomarker is α-fetoprotein (AFP), a surveillance biomarker for hepatocellular carcinoma (HCC) that contains a glycosylation site at asparagine 251. The low diagnostic sensitivity of AFP led researchers to focus on AFP-L3, which has the same sequence as conventional AFP but contains a fucosylated glycan. AFP-L3 has high affinity for Lens culinaris agglutinin (LCA) lectin, prompting many groups to use it for detecting AFP-L3. However, a few studies have identified more effective lectins for fractionating AFP-L3. In this study, we compared the amounts of enriched AFP-L3 with five fucose-specific lectinsLCA, Lotus tetragonolobus lectin (LTL), Ulex europaeus agglutinin I (UEA I), Aleuria aurantia lectin (AAL), and Aspergillus oryzae lectin (AOL)to identify better lectins and improve HCC diagnostic assays using mass spectrometry (MS). Our results indicate that LTL was the most effective lectin for capturing AFP-L3 species, yielding approximately 3-fold more AFP-L3 than LCA from the same pool of HCC serum samples. Thus, we recommend the use of LTL for AFP-L3 assays, given its potential to improve the diagnostic sensitivity in patients having limited results by conventional LCA assay. The MS data have been deposited to the PeptideAtlas (PASS01752).

Quantitative analysis of fucosylated glycoproteins by immobilized lectin-affinity fluorescent labeling

“…We used a combination or one of AAL, LCA, UEA-I lectins, which can bind a1,6, a1,2, and a1,3 linked fucoses. 32,53 Thus, this method can quantify glycoprotein with stages with hyperfucosylation of lung metastatic saliva (Fig. 6C).…”

“…We used a combination or one of AAL, LCA, UEA-I lectins, which can bind α1,6, α1,2, and α1,3 linked fucoses. 32,53 Thus, this method can quantify glycoprotein with core or antenna fucoses. IgG is one of the most abundant fucosylated glycoproteins in serum, and its abundance correlates with humoral immune status.…”

“…In this study, we used a combination of lectins (AAL, UEA-I, and LCA) for quantitative analysis of salivary protein fucosylation. The combination of AAL and LCA enhanced the identification of α1,6 fucosylated glycoproteins for better quantification, 32 while UEA-I enriched for antennary α1,2 fucosylated glycoproteins in tumor cells. 33 Lectins are first covalently bound to amine-reactive beads, while proteins extracted from saliva are labeled with fluorescein tags ( Fig.…”

Glycosylation Profiling of the Neoplastic Biomarker Alpha Fetoprotein through Intact Mass Protein Analysis

“…Several liquid chromatography with tandem mass spectrometry (LC–MS/MS) methods have been developed to profile and identify different glycoforms of AFP, which are based on quantification of AFP-specific glycopeptides through digestion of proteins enriched from plasma or serum samples. ,, These methods utilize glycopeptides representing the AFP glycoforms without core fucosylation (AFP-L1) and with core fucosylation (AFP-L3) to calculate the relative abundance of AFP-L3 glycoforms (AFP-L3%). Other studies also utilized enzymes and chemical treatment to release glycans from AFP and identified the glycan structure by electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) coupled with MS. ,, During MS analysis, glycans and glycopeptides require different collision energies for structure and sequence identification based on fragmentation.…”

Characterization of glycosylation regulator-mediated glycosylation modification patterns and tumor microenvironment infiltration in hepatocellular carcinoma

“…Glycosylation of LAMP2 and SV40 serves as a protective mechanism against HCC (Pousset et al, 1997;Chiu et al, 2022). Glycosylation of HCC mostly occurs in oncogenes such as AFP, AACT, and MDR, which can be used as risk factors for HCC (Ledoux et al, 2003;Lee et al, 2022). Glycosylation of these genes is not only involved in the molecular mechanism of HCC progression but is also closely related to tumor drug resistance.…”