MSACL 2016 EU Abstract

Uncovering Target Glycoprotein Biosignatures Using a One-Pot Dual Nanoprobe Mass Spectrometry Assay

Mira Anne dela Rosa (Presenter)
Institute of Chemistry, Academia Sinica

Authorship: Mira Anne C. dela Rosa,(1,2,3) Wei-Chun Chen,(3) Yi-Ju Chen,(3) Rofeamor P. Obena,(3) Chih-Hsiang Chang,(3) Rey Y. Capangpangan,(3) Tung-Hung Su,(4) Chi-Ling Chen,(4) Pei-Jer Chen(4) and Yu-Ju Chen(1,3)
(1) Department of Chemistry, National Taiwan University, (2) Nano Science and Technology Program, Taiwan International Graduate Program, Academia Sinica (2), (3) Institute of Chemistry, Academia Sinica, (4) Graduate Institute of Clinical Medicine, National Taiwan University

Short Abstract

We designed a one-pot enrichment strategy to achieve sequential target glycoprotein and glycopeptide purification. By employing dual nanoprobes with divergent separation properties and functionality, a multi-step assay is achieved in a single container ("one-pot"). As demonstrated on alpha-fetoprotein (AFP), the assay has high purification specificity with 2-fold more glycopeptides compared to non-one-pot method. By targeted MS analysis (MRM-MS) of the non-glycopeptides, the assay can quantify low abundant AFP expression (0.5 ng) with good correlation with conventional ELISA method (Pearson's r=0.987). Furthermore, we present the first study revealing AFP glycopeptide patterns of individual liver cancer patients, comprised of 59 heterogeneous glycoforms of bi- and triantennary, core and terminal fucosylation, and mono- to tri-sialylation.

Long Abstract

Although quantification of protein biomarker concentration in biofluid is currently used to diagnose disease, their utility is limited because many biomarkers do not have sufficient specificity and sensitivity to diagnose the disease. Alternatively, aberrations in protein post-translational modifications (PTMs) are increasingly being studied because of their close association with cancer and other diseases. Changes on the glycan structure, site occupancy and glycoprotein concentration can be markers of disease development and progression. Mass spectrometry (MS) is a powerful tool to analyze glycosylation in complex mixtures. However, it is imperative to reduce the complexity of biofluids because the low abundance of the target glycoprotein (pg/mL-low ng/mL) limits their efficient analysis. To further characterize the high degree of glycan structure diversity, afterwards, enrichment of glycopeptides has to be employed to obtain information on glycan structure and site occupancy. As a result, intact glycosylated peptides analysis requires multiple steps and longer time to ensure isolation specificity at both protein and peptide levels. To circumvent these issues, we designed a one-pot enrichment strategy to achieve sequential target glycoprotein and glycopeptide purification. By employing dual nanoprobes with divergent separation properties and functionality, a multi-step assay is achieved in a single container ("one-pot"). Combined with liquid chromatography-tandem MS (LC-MS/MS) analysis for quantification and glycosylation profiling, we demonstrated this strategy to alpha-fetoprotein (AFP), a biomarker for liver cancer, as a proof-of-concept. The assay has high purification specificity with 2-fold more glycopeptides compared to non-one-pot method. By targeted MS analysis (MRM-MS) of the non-glycopeptides, the assay can quantify low abundant AFP expression (0.5 ng) with good correlation with conventional ELISA method (Pearson's r=0.987). Furthermore, we present the first study revealing AFP glycopeptide patterns of individual liver cancer patients, comprised of 59 heterogeneous glycoforms of bi- and triantennary, core and terminal fucosylation, and mono- to tri-sialylation. Thus, through this one-pot assay, a comprehensive target protein profile comprised of protein expression level and its glycosylation pattern was achieved in simple sample preparation protocol with high sensitivity, reduced analysis time and minute starting material. This assay can be extended to other PTM-proteins by conjugation of other affinity ligands on the nanoprobe.


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