Advances in the proteomic investigation of the cell secretome

Abstract

Studies of the cell secretome have greatly increased in recent years owing to improvements in proteomic platforms, mass spectrometry instrumentation and to the increased interaction between analytical chemists, biologists and clinicians. Several secretome studies have been implemented in different areas of research, leading to the generation of a valuable secretome catalogs. Secreted proteins continue to be an important source of biomarkers and therapeutic target discovery and are equally valuable in the field of microbiology. Several discoveries have been achieved in vitro using cell culture systems, ex vivo using human tissue specimens and in vivo using animal models. In this review, some of the most recent advances in secretome studies and the fields that have benefited the most from this evolving technology are highlighted.

Figure 1. Generic in vitro secretome profiling flow chart

Typically, cells are grown to 80 or 100% confluence in serum-supplemented media, then washed (up to six times with serum-free medium or phosphate-buffered saline) and incubated for an additional 12–24 h in serum-free media. Conditioned medium containing secreted proteins is collected, centrifuged at 300 g to remove floating cells and gross cell debris, then further passed through a 0.22-µm filter to remove small debris. Conditioned medium is then concentrated up to 200-fold using a centrifugal filtration device (usually with a 3-kDa molecular weight cutoff). Protein concentration is measured and usually 50–100 µg of total protein is fractionated by SDS-PAGE. The resulting lane is sliced into 30–35 bands for in-gel digestion and LC-MS/MS analysis. Resulting data are searched against the desired protein database using a suitable search engine (such as Sequest or Mascot). The obtained list is then further curated for protein subcellular localization using the Uniprot knowledgebase [104]. Unclassified proteins are checked for potential secretion using the SignalP server [105]. Other proteins can be searched manually for subcellular localization in the literature or exosome databases.

Figure 2. Numbers and disease distribution of secretome publications

(A) The number of articles describing secretome studies has been on the rise since the year 2000, when only one article was published, to the year 2011 when more than 570 articles were published. (B) A large number of secretome studies have dealt with cancer (26%) and microbiology (14%). Veterinary medicine, including murine models, constituted 13.1%, toxicology 7.7%, diabetes 3.3%, infectious diseases 1.5% and AIDS 1.4% of the published articles. Data were obtained by using the NCBI Pubmed Central database. Limits were set to human and animals and queried searching the word ‘secretome’ and manually verified for inclusion. To generate disease-specific secretome studies, disease type (e.g., cancer) was included in the limits.

Figure 3. Pie chart distribution showing secretome profiling studies by cancer type

Data were obtained by using the NCBI Pubmed Central with a query of keywords ‘secretome’ and cancer type.