Quantitative Secretome Analysis of Activated Jurkat Cells Using Click Chemistry-Based Enrichment of Secreted Glycoproteins

Quantitative
secretome analyses are a high-performance tool for
the discovery of physiological and pathophysiological changes in cellular
processes. However, serum supplements in cell culture media limit
secretome analyses, but serum depletion often leads to cell starvation
and consequently biased results. To overcome these limiting factors,
we investigated a model of T cell activation (Jurkat cells) and performed
an approach for the selective enrichment of secreted proteins from
conditioned medium utilizing metabolic marking of newly synthesized
glycoproteins. Marked glycoproteins were labeled via bioorthogonal
click chemistry and isolated by affinity purification. We assessed
two labeling compounds conjugated with either biotin or desthiobiotin
and the respective secretome fractions. 356 proteins were quantified
using the biotin probe and 463 using desthiobiotin. 59 proteins were
found differentially abundant (adjusted p-value ≤0.05,
absolute fold change ≥1.5) between inactive and activated T
cells using the biotin method and 86 using the desthiobiotin approach,
with 31 mutual proteins cross-verified by independent experiments.
Moreover, we analyzed the cellular proteome of the same model to demonstrate
the benefit of secretome analyses and provide comprehensive data sets
of both. 336 proteins (61.3%) were quantified exclusively in the secretome.
Data are available via ProteomeXchange with identifier PXD004280.