Heeyoun Hwang

Integrated GlycoProteome Analyzer (I-GPA) for Automated Identification and Quantitation of Site-Specific N-Glycosylation.

Human glycoproteins exhibit enormous heterogeneity at each N-glycosite, but few studies have attempted to globally characterize the site-specific structural features. We have developed Integrated GlycoProteome Analyzer (I-GPA) including mapping system for complex N-glycoproteomes, which combines methods for tandem mass spectrometry with a database search and algorithmic suite. Using an N-glycopeptide database that we constructed, we created novel scoring algorithms with decoy glycopeptides, where 95 N-glycopeptides from standard α1-acid glycoprotein were identified with 0% false positives, giving the same results as manual validation. Additionally automated label-free quantitation method was first developed that utilizes the combined intensity of top three isotope peaks at three highest MS spectral points. The efficiency of I-GPA was demonstrated by automatically identifying 619 site-specific N-glycopeptides with FDR ≤ 1%, and simultaneously quantifying 598 N-glycopeptides, from human plasma samples that are known to contain highly glycosylated proteins. Thus, I-GPA platform could make a major breakthrough in high-throughput mapping of complex N-glycoproteomes, which can be applied to biomarker discovery and ongoing global human proteome project.

In-depth analysis of site-specific N-glycosylation in vitronectin from human plasma by tandem mass spectrometry with immunoprecipitation

The characterization of site-specific microheterogeneity in glycoprotein is very important for understanding cell biology and disease processes. Vitronectin is well known to be a multifunctional glycoprotein in the blood and the extracellular matrix, which is related to hepatocellular carcinoma (HCC). Here, we systematically analyzed the site-specific N-glycopeptides of vitronectin in human plasma by tandem mass spectrometry combined with immunoprecipitation and hydrophilic interaction liquid chromatography (HILIC) enrichment. Vitronectin was purified with immunoprecipitation by monoclonal antibody from plasma and digested to tryptic N-glycopeptides.Then, enrichment with HILIC materials was used and followed by analysis with nano-LC/MS/MS. The sequences of N-glycopeptides were identified from the mass spectra by high-energy C-trap dissociation (HCD) and collision-induced dissociation (CID). In HCD mode, oxonium ions were used for recognizing glycopeptides and y ions for sequencing the peptide backbone. In CID mode, Y ions were used for characterizing their glycoforms. As a result, a total of 17 site-specific N-glycopeptides were completely identified in all of the three N-glycosylation sites of vitronectin in human plasma, including 12 N-glycopeptides first reported. Finally, we specifically found that three hybrid and four complex glycopeptides of triantennary forms with outer fucosylation increased in HCC human plasma.

Chromosome-Based Proteomic Study for Identifying Novel Protein Variants from Human Hippocampal Tissue Using Customized neXtProt and GENCODE Databases

The goal of the Chromosome-Centric Human Proteome Project (C-HPP) is to fully provide proteomic information from each human chromosome, including novel proteoforms, such as novel protein-coding variants expressed from noncoding genomic regions, alternative splicing variants (ASVs), and single amino acid variants (SAAVs). In the 144 LC/MS/MS raw files from human hippocampal tissues of control, epilepsy, and Alzheimers disease, we identified the novel proteoforms with a workflow including integrated proteomic pipeline using three different search engines, MASCOT, SEQUEST, and MS-GF+. With a <1% false discovery rate (FDR) at the protein level, the 11 detected peptides mapped to four translated long noncoding RNA variants against the customized databases of GENCODE lncRNA, which also mapped to coding-proteins at different chromosomal sites. We also identified four novel ASVs against the customized databases of GENCODE transcript. The target peptides from the variants were validated by tandem MS fragmentation pattern from their corresponding synthetic peptides. Additionally, a total of 128 SAAVs paired with their wild-type peptides were identified with FDR <1% at the peptide level using a customized database from neXtProt including nonsynonymous single nucleotide polymorphism (nsSNP) information. Among these results, several novel variants related in neuro-degenerative disease were identified using the workflow that could be applicable to C-HPP studies. All raw files used in this study were deposited in ProteomeXchange (PXD000395).

Analysis of fucosylation in liver-secreted N-glycoproteins from human hepatocellular carcinoma plasma using liquid chromatography with tandem mass spectrometry

AbstractFucosylation of N-glycoproteins has been implicated in various diseases, such as hepatocellular carcinoma (HCC). However, few studies have performed site-specific analysis of fucosylation in liver-secreted proteins. In this study, we characterized the fucosylation patterns of liver-secreted proteins in HCC plasma using a workflow to identify site-specific N-glycoproteins, where characteristic B- and/or Y-ion series with and without fucose in collision-induced dissociation were used in tandem mass spectrometry. In total, 71 fucosylated N-glycopeptides from 13 major liver-secreted proteins in human plasma were globally identified by LC-MS/MS. Additionally, 37 fucosylated N-glycopeptides were newly identified from nine liver-secreted proteins, including alpha-1-antichymotrypsin, alpha-1-antitrypsin, alpha-2-HS-glycoprotein, ceruloplasmin, alpha-1-acid glycoprotein 1/2, alpha-2-macroglobulin, serotransferrin, and beta-2-glycoprotein 1. Of the fucosylated N-glycopeptides, bi- and tri-antennary glycoforms were the most common ones identified in liver-secreted proteins from HCC plasma. Therefore, we suggest that this analytical method is effective for characterizing fucosylation in liver-secreted proteins. Graphical abstractA global map of fucosylated and non-fucosylated glycopeptides from 13 liver-secreted glycoproteins in hepatocellular carcinoma plasma

Direct Monitoring of Fucosylated Glycopeptides of Alpha-Fetoprotein in Human Serum for Early Hepatocellular Carcinoma by Liquid Chromatography-Tandem Mass Spectrometry with Immunoprecipitation

Purpose: Alpha‐fetoprotein (AFP) is a widely used serological marker that is associated with hepatocellular carcinoma (HCC). Although the level of AFP is increased in HCC, its sensitivity for diagnosis is poor because AFP levels are also increased in liver diseases. Changes in glycoform, especially fucosylation, have been reported to be associated with the development of HCC.

Identification of Missing Proteins in Human Olfactory Epithelial Tissue by Liquid Chromatography–Tandem Mass Spectrometry

We performed proteomic analyses of human olfactory epithelial tissue to identify missing proteins using liquid chromatography-tandem mass spectrometry. Using a next-generation proteomic pipeline with a < 1.0% false discovery rate at the peptide and protein levels, we identified 3731 proteins, among which five were missing proteins (P0C7M7, P46721, P59826, Q658L1, and Q8N434). We validated the identified missing proteins using the corresponding synthetic peptides. No olfactory receptor (OR) proteins were detected in olfactory tissue, suggesting that detection of ORs would be very difficult. We also identified 49 and 50 alternative splicing variants mapped at the neXtProt and GENCODE databases, respectively, and 2000 additional single amino acid variants. This data set is available at the ProteomeXchange consortium via PRIDE repository (PXD010025).