This approach combines Gene Ontology (GO) annotation and WoLF PSORT [1] localization prediction to determine proteins that are expected to be part of the cellular secretome, i.e. proteins either secreted by cells or shed from the cellular surface. For this workflow, we chose to include all proteins annotated or predicted as lysosomal proteins. Lysosomal proteins are routinely secreted by malignant and non-malignant cells in high amounts, due to "leakiness" of the mannose-6-phosphate receptor pathway [2,3]. Furthermore, we chose to exclude proteins annotated as being part of extracellular organelles, e.g. exosomes. While exosomes are secreted by malignant and non-malignant cells, exosomal proteins are expected in the secretome at very low amounts, if not especially enriched for.
[1] P. Horton, K.J. Park, T. Obayashi, N. Fujita, H. Harada, C.J. Adams-Collier, et al., WoLF PSORT: Protein localization predictor, Nucleic Acids Res. 35 (2007) 585–587. doi:10.1093/nar/gkm259.
[2] B. Schröder, C. Wrocklage, A. Hasilik, P. Saftig, The proteome of lysosomes., Proteomics. 10 (2010) 4053–76. doi:10.1002/pmic.201000196.
[3] J. Reiser, B. Adair, T. Reinheckel, Specialized roles for cysteine cathepsins in health and disease, J. Clin. Invest. 120 (2010) 3421–3431. doi:10.1172/JCI42918.
The workflow needs three input files:
- A tabular file, the first column containing uniprot accession numbers of the proteins of interest.
- The complete uniprot GO database for the organism of interest.
- The complete GO Open Biomedical Ontology (OBO), i.e. "GO term tree", accessible at http://purl.obolibrary.org/obo/go/go.obo.
As an example dataset for input 1, you can use a list of human proteins, identified by LC-MS/MS in the cellular supernatant of MDA-MB-231 cells.
If you use this workflow directly, or a derivative of it, in work leading to a scientific publication, please cite:
F.C. Sigloch, J.D. Knopf, J. Weißer, A. Gomez-Auli, M.L. Biniossek, A. Petrera, et al., Proteomic analysis of silenced cathepsin B expression suggests non-proteolytic cathepsin B functionality, Biochim. Biophys. Acta - Mol. Cell Res. 1863 (2016) 2700–2709. doi:10.1016/j.bbamcr.2016.08.005. https://www.ncbi.nlm.nih.gov/pubmed/27526672
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