Reference glycan structure libraries of primary human cardiomyocytes and pluripotent stem cell-derived cardiomyocytes reveal cell-type and culture stage-specific glycan phenotypes
Ashwood C, Waas M, Weerasekera R, Gundry RL. Reference glycan structure libraries of primary human cardiomyocytes and pluripotent stem cell-derived cardiomyocytes reveal cell-type and culture stage-specific glycan phenotypes. Journal of Molecular and Cellular Cardiology. 2020 Jan 21.
- Organism: Homo sapiens
- Instrument: LTQ Orbitrap Velos
N-glycan, glycomics, O-glycan, cardiac, heart, stem-cell
Lab head: Rebekah Gundry
Submitter: Christopher Ashwood
Cell surface glycoproteins play critical roles in maintaining cardiac structure and function in health and disease and the glycan-moiety attached to the protein is critical for proper protein folding, stability and signaling. However, despite mounting evidence that glycan structures are key modulators of heart function and must be considered when developing cardiac biomarkers, we currently do not have a comprehensive view of the glycans present in the normal human heart. In the current study, we used porous graphitized carbon liquid chromatography interfaced with mass spectrometry (PGC-LC-MS) to generate glycan structure libraries for primary human heart tissue homogenate, cardiomyocytes (CM) enriched from human heart tissue, and human induced pluripotent stem cell derived CM (hiPSC-CM). Altogether, we established the first reference structure libraries of the cardiac glycome containing 265 N- and O-glycans. These data will benefit future functional glycomics studies by informing the genes, proteins and metabolites responsible for the end-result structures and the glycan isomer libraries defined in this study are necessary for interpretation of future intact glycopeptide data. The cell-type specific glycan differences observed here will support the generation of cell-type specific protein glycoform maps of the human heart, thereby promoting the development of cell type-specific targeting strategies. Moreover, by comparing primary CM to hiPSC-CM collected during 20-100 days of differentiation, dynamic changes in the glycan profile throughout in vitro differentiation and differences between primary and hiPSC-CM are revealed. These observations are an important complement to current genomic, transcriptomic, and proteomic profiling and reveal new considerations for the use and interpretation of hiPSC-CM models for studies of human development, disease, and drug testing. Finally, these data are expected to support future regenerative medicine efforts by informing targets for evaluating the immunogenic potential of hiPSC-CM and harnessing differences between immature, proliferative hiPSC-CM and adult primary CM.
Samples were batched by replicate and samples within each batch were randomized prior to injection. A internal standard only sample was analyzed between each replicate block to verify no carry-over. PGC-LC-ESI-MS/MS experiments were performed using a nanoLC-2D high performance liquid chromatography system (Eksigent, CA, USA) interfaced with an LTQ Orbitrap Velos hybrid mass spectrometer (Thermo Fisher Scientific). Released glycans were separated on a PGC-LC column (3 μm, 100 mm × 0.18 mm, Hypercarb, Thermo Fisher Scientific) maintained at 80°C for N-glycans and 40°C for O-glycans. To enhance ionization and improve detection of low abundance glycans, post-column make-up flow consisting of 100% methanol was utilized. DDA acquisition.
Released and reduced N- and O-glycans from human heart tissue homogenate, cardiomyocytes enriched from human heart tissue and stem-cell derived CM between 20-100 days of differentiation.
Created on 9/5/19, 10:05 PM