Khoo's Lab - Site-specific Glycosylation

On site-specific glycosylation, our studies of coronavirus spike proteins established MS-based site-specific glycosylation analysis as an integral component of structural glycobiology. Our work (Kuo et al., Glycobiology, 2022), published a year after we obtained the SARS-CoV-2 spike protein trimeric ectodomain construct, is the first to report distinct evolutionary site-specific changes in spike protein glycosylation associated with D614G and Alpha variants, underscoring that the glycan shield is dynamic and must be resolved at individual glycosylation sites. Extending to other coronaviruses, our glycosylation analyses were integrated with cryo-ET/cryo-EM to define alphacoronavirus spike architecture and dynamics (Huang et al., Nature Comm, 2022), incorporated into computational approaches for constructing realistic glycoprotein structures using experimentally informed glycan conformer libraries and steric constraints (Tsai et al., Cell, 2024), and applied in structural analysis of human coronavirus 229E host recognition (Tsai et al., Nature Comm, 2025). These studies provided a demanding test bed for critically evaluating the differences among glycopeptide-analysis software in search strategy, scoring, and glycan assignment. By exploiting their complementary strengths, we developed a practical N-glycopeptide MS/MS workflow that improves confidence in site-specific identification and glycan assignment (Kuo et al., Proteomics, 2023).

The same analytical-to-structural progression was taken further in our study of receptor-type protein tyrosine phosphatase alpha (PTPRA). What began as an effort to define its site-specific glycosylation revealed an exceptionally dense mixture of N- and O-glycans that pushed current bottom-up MS approaches close to their practical limit. Through advanced LC-MS/MS and complementary proteolysis, including O-glycoproteases, we achieved extensive site-resolved characterization of this highly heterogeneous ectodomain. The work then expanded into a collaboration involving four IBC laboratories and Japanese investigators, integrating MS with high-speed AFM, biophysical analysis and molecular modeling. This showed that dense N- and O-glycosylation forms a protective molecular shield and constrains the dimensions and dynamics of the intrinsically disordered PTPRA ectodomain (Chien et al., JACS Au, 2023).

Kuo CW, Yang TJ, Chien YC, Yu PY, Hsu SD, and Khoo KH (2022) Distinct shifts in site-specific glycosylation pattern of SARS-CoV-2 spike proteins associated with arising mutations in the D614G and Alpha variants. Glycobiology 32, 60-72. https://doi.org/10.1093/glycob/cwab102
Huang CY, Draczkowski P, Wang YS, Chang CY, Chien YC, Cheng YH, Wu YM, Wang CH, Chang YC, Chang YC, Yang TJ, Tsai YX, Khoo KH, Chang HW, and Hsu SD (2022) In situ structure and dynamics of an alphacoronavirus spike protein by cryo-ET and cryo-EM. Nat Commun 13, 4877. https://doi.org/10.1038/s41467-022-32588-3
Tsai YX, Chang NE, Reuter K, Chang HT, Yang TJ, von Bulow S, Sehrawat V, Zerrouki N, Tuffery M, Gecht M, Grothaus IL, Colombi Ciacchi L, Wang YS, Hsu MF, Khoo KH, Hummer G, Hsu SD, Hanus C, and Sikora M (2024) Rapid simulation of glycoprotein structures by grafting and steric exclusion of glycan conformer libraries. Cell 187, 1296-1311 e26. https://doi.org/10.1016/j.cell.2024.01.034
Kuo CW, Chang NE, Yu PY, Yang TJ, Hsu SD, and Khoo KH* (2023) An N-glycopeptide MS/MS data analysis workflow leveraging two complementary glycoproteomic software tools for more confident identification and assignments. Proteomics, e2300143. https://doi.org/10.1002/pmic.202300143
Chien YC, Wang YS, Sridharan D, Kuo CW, Chien CT, Uchihashi T, Kato K, Angata T, Meng TC, Hsu SD, and Khoo KH* (2023) High Density of N- and O-Glycosylation Shields and Defines the Structural Dynamics of the Intrinsically Disordered Ectodomain of Receptor-type Protein Tyrosine Phosphatase Alpha. JACS Au 3, 1864-1875. https://doi.org/10.1021/jacsau.3c00124

Other representative works:

Glycoproteomic software solutions spotlight glycans. (2021). Khoo KH. Nat Methods 18(12): 1457-1458
ZIC-cHILIC-Based StageTip for Simultaneous Glycopeptide Enrichment and Fractionation toward Large-Scale N-Sialoglycoproteomics. (2021). Chen YJ, Yen TC, Lin YH, Chen YL, Khoo KH and Chen YJ. Anal Chem 93(48): 15931-15940.
Advances toward mapping the full extent of protein site-specific O-GalNAc glycosylation that better reflects underlying glycomic complexity. (2019). Khoo KH. Curr Opin Struct Biol 56: 146-154.
Distinctive and Complementary MS(2) Fragmentation Characteristics for Identification of Sulfated Sialylated N-Glycopeptides by nanoLC-MS/MS Workflow. (2018). Kuo CW, Guu SY and Khoo KH. J Am Soc Mass Spectrom 29(6): 1166-1178
Adapting Data-Independent Acquisition for Mass Spectrometry-Based Protein Site-Specific N-Glycosylation Analysis. (2017). Pan KT, Chen CC, Urlaub H and Khoo KH. Anal Chem 89(8): 4532-4539
Novel LC-MS2 product dependent parallel data acquisition function and data analysis workflow for sequencing and identification of intact glycopeptides. (2014). Wu SW, Pu TH, Viner R and Khoo KH. Anal Chem 86(11): 5478-5486