Abstract
The plasma membrane proteome plays a crucial role in inter- and intracellular signaling, cell survival, and cell identity. As such, it is a prominent target for pharmacological intervention. The relatively low abundance of this subproteome in conjunction with challenging extractability and solubility still hampers its comprehensive analysis. Here, we combined a chemical glycoprotein-tagging strategy with mass spectrometry to enable comprehensive analysis of the cell-surface glycoproteome. To benchmark this workflow and to provide guidance for cell line selection for functional experiments, we generated an inventory of the N-linked cell-surface glycoproteomes of 15 standard laboratory human cell lines and three primary lymphocytic cell types. On average, about 900 plasma membrane and secreted proteins were identified per experiment, including more than 300 transporters and ion channels. Primary cells displayed distinct expression of surface markers and transporters underpinning the importance of carefully validating model cell lines selected for the study of cell surface-mediated processes. To monitor dynamic changes of the cell-surface proteome in a highly multiplexed experiment, we employed an isobaric mass tag-based chemical labeling strategy. This enabled the time-resolved analysis of plasma membrane protein presentation during differentiation of the monocytic suspension cell line THP-1 into macrophage-like adherent cells. Time-dependent changes observed in membrane protein presentation reflect functional remodeling during the phenotypic transition in three distinct phases: rapid surface presentation and secretion of proteins from intracellular pools concurrent with rapid internalization of no longer needed proteins and finally delayed presentation of newly synthesized macrophage markers. Perturbation of this process using marketed receptor tyrosine kinase inhibitors revealed dasatinib to severely compromise macrophage differentiation due to an off-target activity. This finding suggests that dynamic processes can be highly vulnerable to drug treatment and should be monitored more rigorously to identify adverse drug effects.
Highlights
This finding suggests that dynamic processes can be highly vulnerable to drug treatment and should be monitored more rigorously to identify adverse drug effects
Sensitivity, and coverage achieved with both approaches by label-free LC-MS/MS analysis of duplicate experiments with 11 different cell lines/ primary cells using 4-h gradient separation on an Orbitrap QExactive platform
We mapped the cell-surface proteomes of commonly used established cell lines and primary cells at a previously unprecedented depth detecting on average Ͼ800 plasma membrane proteins per cell type and Ͼ2400 distinct cell-surface proteins in 21 different cell systems. These numbers compared very favorably to the 300 proteins per cell type and in total 1492 cell-surface proteins from 41 human cell types detected in a recent publication (18)
Summary
This finding suggests that dynamic processes can be highly vulnerable to drug treatment and should be monitored more rigorously to identify adverse drug effects. For identification of significant proteins regulated during differentiation of THP-1 cells, a modified t test (30) was applied, and data were filtered for log2-transformed relative abundances of Ն2 or ՅϪ2 at least once between 24 and 72 h of the time course with Benjamini-Hochberg corrected p values Ͻ0.01 at the respective time point. Whereas cell surface-enriched samples were analyzed in a single 4-h LC-MS/MS run, the published whole proteome data was based on three replicates each fractionated into six fractions corresponding to 60 h of total analysis time per cell line.
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