Adipogenesis is the process of adipose-derived stem cell (ADSC) proliferation followed by differentiation into mature adipocytes. An integrated glycomics and proteomics approach was undertaken here to compare native adipocytes with their stem cell progenitors and in vitro adipogenic counterparts, to study the mechanisms underpinning adipocyte biology.
Membrane proteins from adipocytes, ADSCs and in vitro-differentiated ADSCs (dADSCs) were separated using SDS-PAGE and the protein bands analysed by shotgun proteomics, with and without prior N-glycan release. The released N-glycans were analysed using porous graphitised carbon (PGC-LC) coupled with ESI-MS/MS, and peptides using RP-LC and ESI-MS/MS.
In the N-glycan analysis, bisecting GlcNAc structures were prominent in adipocytes across all molecular weight bands of the separated membrane proteins, but not on the stem cell or dADSC proteins. Subsequent proteomic analysis of specific PNGase F de-N-glycosylated protein bands identified differential expression of the glycoprotein CD36, which was the major protein in adipocytes.
Traditional global shotgun proteomics analysis of gel-separated bands however showed that CD36, an adipocyte marker that plays a key role in lipid accumulation, did not statistically change in abundance between adipocytes, stem cells and dADSCs. This was likely because the glycosylated CD36 sample had only 28% sequence coverage, compared to 70% when N-glycans were removed prior to trypsin digestion. Western blotting and qPCR confirmed the high CD36 abundance in adipocytes compared to stem cells and dADSCs.
These results demonstrate the crucial role protein de-N-glycosylation plays before standard proteomics analysis for uncovering true abundance levels of key components of the (glyco)proteome. They also show the utility of a multi-omic approach in determining that the molecular composition of in vivo cell differentiation (native adipocytes) is not reflected in the progenitor stem cells or, importantly, in vitro differentiated adipocytes in terms of both protein N-glycan type (i.e. bisecting GlcNAc) and total CD36 protein expression levels.