TY - JOUR
T1 - Glycan node profiling of soluble and membrane glycoproteins in whole cell lysates
AU - Aguilar Díaz de león, Jesús S.
AU - Cruz Villarreal, Jorvani
AU - Kapuruge, Erandi P.
AU - Borges, Chad R.
N1 - Publisher Copyright:
© 2023 Elsevier Inc.
PY - 2023/11/1
Y1 - 2023/11/1
N2 - Glycan node analysis (GNA) is a molecularly bottom-up glycomics technique based on the relative quantification of glycan linkage-specific monosaccharide units (“glycan nodes”). It was originally applied to blood plasma/serum, where it detected and predicted progression, reoccurrence, and survival in different types of cancer. Here, we have adapted this technology to previously inaccessible membrane glycoproteins from cultured cells. The approach is facilitated by methanol/chloroform precipitation of cell lysates and a “liquid phase permethylation” (LPP) procedure. LPP gave better signal-to-noise, yield and precision for most of the glycan nodes from membrane glycoproteins/glycolipids than the conventional solid phase permethylation approach. This GNA approach in cell lysates revealed that specific glycan features such as antennary fucosylation, N-glycan branching, and α2,6-sialylation were elevated in hepatocellular carcinoma (HepG2) cells relative to leukemia cells (THP-1 and K562) and normal donor PBMCs. Additional nodes commonly associated with glycolipids were elevated in the leukemia cells relative to HepG2 cells and PBMCs. Exposure of HepG2 cells to a fucosyltransferase inhibitor resulted in a significant reduction in the relative abundance of 3,4-substituted GlcNAc, which represents antennary fucosylation—providing further proof-of-concept that downregulation of glycosyltransferase activity is detected by shifts in glycan node expression—now detectable in membrane glycoproteins.
AB - Glycan node analysis (GNA) is a molecularly bottom-up glycomics technique based on the relative quantification of glycan linkage-specific monosaccharide units (“glycan nodes”). It was originally applied to blood plasma/serum, where it detected and predicted progression, reoccurrence, and survival in different types of cancer. Here, we have adapted this technology to previously inaccessible membrane glycoproteins from cultured cells. The approach is facilitated by methanol/chloroform precipitation of cell lysates and a “liquid phase permethylation” (LPP) procedure. LPP gave better signal-to-noise, yield and precision for most of the glycan nodes from membrane glycoproteins/glycolipids than the conventional solid phase permethylation approach. This GNA approach in cell lysates revealed that specific glycan features such as antennary fucosylation, N-glycan branching, and α2,6-sialylation were elevated in hepatocellular carcinoma (HepG2) cells relative to leukemia cells (THP-1 and K562) and normal donor PBMCs. Additional nodes commonly associated with glycolipids were elevated in the leukemia cells relative to HepG2 cells and PBMCs. Exposure of HepG2 cells to a fucosyltransferase inhibitor resulted in a significant reduction in the relative abundance of 3,4-substituted GlcNAc, which represents antennary fucosylation—providing further proof-of-concept that downregulation of glycosyltransferase activity is detected by shifts in glycan node expression—now detectable in membrane glycoproteins.
KW - Cell lysate
KW - Glycan nodes
KW - Glycomics
KW - Liquid phase permethylation. solid phase permethylation
KW - Membrane glycoproteins
KW - Permethylation
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U2 - 10.1016/j.ab.2023.115317
DO - 10.1016/j.ab.2023.115317
M3 - Article
C2 - 37699507
AN - SCOPUS:85170639672
SN - 0003-2697
VL - 680
JO - Analytical Biochemistry
JF - Analytical Biochemistry
M1 - 115317
ER -