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

Elevated serum alpha-fetoprotein (AFP) can be observed in liver cirrhosis and hepatocellular carcinoma (HCC). The glycosylation patterns of AFP have been shown to differentiate these conditions, with AFP glycoforms with core fucosylation (AFP-L3) serving as a malignancy risk predictor for HCC. We have developed a method to detect endogenously present AFP proteoforms and to quantify the relative abundance of AFP-L3 glycoforms (AFP-L3%) in serum samples. This method consists of immune enrichment of endogenous AFP, followed by liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) intact protein analysis of AFP. Data are available via ProteomeXchange with identifier PXD038606. Based on the AFP profiles in authentic patient serum samples, we have identified that the frequently observed AFP glycoforms without core fucosylation (AFP-L1) are G2S2 and G2S1, and common AFP-L3 glycoforms are G2FS1 and G2FS2. The intensities of glycoforms in the deconvoluted spectrum are used to quantify AFP-L3% in each sample. The method evaluation included reproducibility, specificity, dilution integrity, and comparison of AFP-L3% with a lectin-binding gel shift electrophoresis (GSE) assay. The AFP-L1 and AFP-L3 proteoforms were reproducibly identified in multiple patient serum samples, resulting in reproducible AFP-L3% quantification. There was considerable agreement between the developed LC-HRMS and commercial GSE methods when quantifying AFP-L3% (Pearson r = 0.63) with a proportional bias.