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Selective enrichment of newly synthesized proteins for quantitative secretome analysis

Nature Biotechnology volume 30, pages 984–990 (2012)Cite this article

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Abstract

Secreted proteins constitute a large and biologically important subset of proteins that are involved in cellular communication, adhesion and migration. Yet secretomes are understudied because of technical limitations in the detection of low-abundance proteins against a background of serum-containing media. Here we introduce a method that combines click chemistry and pulsed stable isotope labeling with amino acids in cell culture to selectively enrich and quantify secreted proteins. The combination of these two labeling approaches allows cells to be studied irrespective of the complexity of the background proteins. We provide an in-depth and differential secretome analysis of various cell lines and primary cells, quantifying secreted factors, including cytokines, chemokines and growth factors. In addition, we reveal that serum starvation has a marked effect on secretome composition. We also analyze the kinetics of protein secretion by macrophages in response to lipopolysaccharides.

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Figure 1: Quantitative analysis of cell secretomes.
Figure 2: Comparison of secretomes between cell types and treatments.
Figure 3: Interaction network of differentially secreted proteins from PC3 and WPMY-1 cells highlighting clusters of functionally related proteins.
Figure 4: Comparison of the secretomes of primary hepatocytes (PHC) and hepatoma cell lines (Hepa1-6 and Hepa1c1).
Figure 5: Kinetics of protein secretion from LPS-stimulated mouse macrophages.
Figure 6: Compendium of secreted proteins identified across all experiments in this study.

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Acknowledgements

We gratefully acknowledge the EMBL Proteomics Core Facility for expert technical assistance. This study was supported by the Netherlands Organisation for Scientific Research (NWO).

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Authors and Affiliations

  1. EMBL, Genome Biology Unit, Heidelberg, Germany

    Katrin Eichelbaum, Markus Winter & Jeroen Krijgsveld

  2. Joint Division Molecular Metabolic Control, DKFZ-ZMBH Alliance, Center for Molecular Biology (ZMBH), University Hospital and German Cancer Research Center (DKFZ), Heidelberg, Germany

    Mauricio Berriel Diaz & Stephan Herzig

Authors
  1. Katrin Eichelbaum
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  2. Markus Winter
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  3. Mauricio Berriel Diaz
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  4. Stephan Herzig
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Contributions

K.E. and J.K. designed the study. K.E., M.W. and M.B.D. performed cell culture experiments with supervision from S.H. K.E. performed biochemical and mass spectrometric experiments. K.E. analyzed the data with input from J.K. K.E. and J.K. wrote the paper with input from all other authors.

Corresponding author

Correspondence to Jeroen Krijgsveld.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 (PDF 1147 kb)

Supplementary Table 1

Secretome analysis of PC3 and WPMY-1 cells (XLSX 345 kb)

Supplementary Table 2

Secretome analysis of primary hepatocytes and hepatoma cell lines Hepa1-6 and Hepa1c1 (XLSX 831 kb)

Supplementary Table 3

Secretome analysis of LPS-stimulated mouse macrophages (XLSX 169 kb)

Supplementary Table 4

Compendium of human secreted proteins (XLSX 2006 kb)

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Eichelbaum, K., Winter, M., Diaz, M. et al. Selective enrichment of newly synthesized proteins for quantitative secretome analysis. Nat Biotechnol 30, 984–990 (2012). https://doi.org/10.1038/nbt.2356

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