One-Pot Two-Nanoprobe Assay Uncovers Targeted Glycoprotein Biosignature.
We report a one-pot two-nanoprobe approach coupled to mass spectrometry for simultaneous quantification and post-translational modification (PTM) profiling of targeted protein in biofluid. Using N-glycoprotein as model, the assay employs two nanoprobes, antibody-conjugated SiO2nanoparticles and lectin-conjugated magnetic Fe3O4nanoparticles, to achieve target glycoprotein isolation from biofluid and subsequent glycopeptide enrichment in a single tube. As demonstrated on α-fetoprotein (AFP), a serum biomarker for hepatocellular carcinoma (HCC), the assay has high purification specificity (20 glycopeptides) with 2-fold and 10-fold superior total glycopeptide intensity compared to non-one-pot method (9 glycopeptides) or without enrichment (6 glycopeptides), respectively. By multiple reaction monitoring mass spectrometry (MRM-MS) analysis of the nonglycopeptides, 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 signatures of individual HCC patients, comprised of 23 heterogeneous glycoforms of bi- and triantennary, core and terminal fucosylation, and mono- to trisialylation. In addition to 12 novel AFP glycoforms, our quantification result uncovers five abundant glycoforms in HCC, including 3 core-fucosylated (CF) forms. These identified CF forms may be evaluated in future studies as potential targets in a glycopeptide biomarker panel to further improve accuracy of conventional AFP-L3 tests. Through this one-pot assay, a comprehensive target protein profile comprised of protein expression and glycosylation pattern was achieved in simple protocol with high sensitivity, reduced analysis time, and minute starting material. This assay can be extended to other PTM biosignatures by conjugation of other affinity ligands on the nanoprobe.
Mira Anne C
MA
Dela Rosa
Department of Chemistry, National Taiwan Normal University , Taipei, Taiwan.
Wei-Chun
WC
Chen
Yi-Ju
YJ
Chen
Rofeamor P
RP
Obena
Chih-Hsiang
CH
Chang
Caraga State University , Butuan City, Philippines.
Rey Y
RY
Capangpangan
Tung-Hung
TH
Su
Chi-Ling
CL
Chen
Pei-Jer
PJ
Chen
Yu-Ju
0000-0002-3178-6697
YJ
Chen
D000906:Antibodies / Q000737:chemistry / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000097:blood* / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000097:blood*; D002853:Chromatography, Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000097:blood* / Q000378:metabolism; D006801:Humans; D037102:Lectins / Q000737:chemistry / Q000378:metabolism; D008113:Liver Neoplasms / Q000097:blood*; D058185:Magnetite Nanoparticles / Q000737:chemistry; D053758:Nanoparticles / Q000737:chemistry*; D011499:Protein Processing, Post-Translational; D012822:Silicon Dioxide / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry
D000906:Antibodies; D014408:Biomarkers, Tumor; D006023:Glycoproteins; D037102:Lectins; D058185:Magnetite Nanoparticles; D012822:Silicon Dioxide