Glycan/Protein-Stable Isotope Labeling in Cell Culture for Enabling Concurrent Quantitative Glycomics/Proteomics/Glycoproteomics

The
complexity and heterogeneity of protein glycosylation present
an analytical challenge to the studies of characterization and quantitation.
Various LC–MS-based quantitation strategies have emerged in
recent decades. Metabolic stable isotope labeling has been developed
to enhance the accurate LC/MS-based quantitation between different
cell lines. Stable isotope labeling by amino acids in a cell culture
(SILAC) is the most widely used metabolic labeling method in proteomic
analysis. However, it can only label the peptide backbone and is thus
limited in glycomic studies. Here, we present a metabolic isotope
labeling strategy, named GlyProSILC (Glycan Protein Stable Isotope Labeling
in Cell Culture), that can label both the glycan motif
and peptide backbone from the same batch of cells. It was performed
by feeding cells with a heavy medium containing amide-15N-glutamine, 13C6-arginine (Arg6), and 13C6-15N2-lysine
(Lys8). No significant change of cell line metabolism after
GlyProSILC labeling was observed based on transcriptomic, glycomic,
and proteomic data. The labeling conditions, labeling efficiency,
and quantitation accuracy were investigated. After quantitation correction,
we simultaneously quantified 62 N-glycans, 574 proteins, and 344 glycopeptides
using the same batch of mixed 231BR/231 cell lines. So far, GlyProSILC
provides an accurate and effective quantitation approach for glycomics,
proteomics, and glycoproteomics in a cell culture system.