Enhancing Comprehensive Analysis of Secreted Glycoproteins from Cultured Cells without Serum Starvation

Abstract

Glycoproteins secreted by cells play essential roles in the regulation of extracellular activities. Secreted glycoproteins are often reflective of cellular status, and thus glycoproteins from easily accessible bodily fluids can serve as excellent biomarkers for disease detection. Cultured cells have been extensively employed as models in the research fields of biology and biomedicine, and global analysis of glycoproteins secreted from these cells provides insights into cellular activities and glycoprotein functions. However, comprehensive identification and quantification of secreted glycoproteins is a daunting task because of their low abundances compared with the high-abundance serum proteins required for cell growth and proliferation. Several studies employed serum-free media to analyze secreted proteins, but it has been shown that serum starvation, even for a short period of time, can alter protein secretion. To overcome these issues, we developed a method to globally characterize secreted glycoproteins and their N-glycosylation sites from cultured cells by combining selective enrichment of secreted glycoproteins with a boosting approach. The results demonstrated the importance of the boosting sample selection and the boostingto-sample ratio for improving the coverage of secreted glycoproteins. The method was applied to globally quantify secreted glycoproteins from THP-1 monocytes and macrophages in response to lipopolysaccharides (LPS) and from Hep G2 cells treated with TGF-β without serum starvation. We found differentially secreted glycoproteins in these model systems that showed the cellular response to the immune activation or the epithelial-to-mesenchymal transition. Benefiting from the selective enrichment and the signal enhancement of low-abundance secreted glycoproteins, this method can be extensively applied to study secreted glycoproteins without serum starvation, which will provide a better understanding of protein secretion and cellular activity.

Spatial and temporal proteomics reveals the distinct distributions and dynamics of O-GlcNAcylated proteins

“…We designed a method integrating metabolic labeling, bioorthogonal chemistry, and multiplexed proteomics to systematically quantify the distribution of O-GlcNAcylated proteins in the nucleus and the cytoplasm ( Figure 1A ). The integration of metabolic labeling and bioorthogonal chemistry is very powerful to study protein glycosylation ( Agatemor et al, 2019 ; Mahal et al, 1997 ; Qin et al, 2017 ; Suttapitugsakul et al, 2019 , 2021a , 2021b ; Vocadlo et al, 2003 ; Woo et al, 2015 ). Glycoproteins were metabolically labeled with N -azidoacetylgalactosamine-tetraacetylated (Ac 4 GalNAz) ( Boyce et al, 2011 ; Xiao et al, 2016 ), and labeled glycoproteins with the small azide group allowed for their selective enrichment through bioorthogonal chemistry ( Hong et al, 2009 ).…”

Quantitative Structural Proteomics Unveils the Conformational Changes of Proteins under the Endoplasmic Reticulum Stress

“…The experimental design is displayed in Figure A. A fully heavy isotope-labeled sample served as a boosting source to assist in the identification of low-abundant newly synthesized proteins. , About 3000 pre-existing proteins were quantified with or without the Tm treatment, while 1783 newly synthesized proteins were quantified in the control samples and 879 proteins in the Tm-treated samples (Figures B, S10, and Table S6). Fewer proteins quantified with the inhibition of N-glycosylation were expected because the decreased overall protein synthesis and the enhanced protein degradation are common cellular responses to relieve the protein unfolding stress.…”

A streamlined tandem tip-based workflow for sensitive nanoscale phosphoproteomics

“…This boosting strategy has recently been demonstrated for effective single-cell proteomics analysis 1, 15, 17 . It has also been applied to study low abundant PTMs such as phosphotyrosine peptides 19 or secreted glycoproteins 49 . When integrated with our streamlined tandem tip-based method, the boosting strategy enabled precise quantification of ∼600 phosphopeptides from 100 sorted cells (Figure 6) as well as >1,500 phosphopeptides from 10 ng of cell lysate digests (∼100 cells; Figure 5).…”