to burst cells, releasing intracellular proteins. In addition, incubation time and conditions must be optimized so that the metabolic stress that can be induced by the lack of nutrients in SFM does not affect secretomic analysis. Low concentration Some proteins are secreted in low abundance and then diluted further in the cell culture medium or body fluid, making these proteins difficult to detect and analyze. Concentration methods like TCA precipitation can be used as well as highly sensitive methods like antibody microarrays that can detect even single molecules of a protein. Relevance of in vitro studies Many secretomic studies are conducted in vitro with cell culture methods, but it is unclear whether the same proteins are secreted in vivo. More and more studies, especially those looking at the cancer secretome, are using in vivo methods to confirm the relevance of the results obtained in vitro. For example, proximal biological fluids can be collected adjacent to a tumor in order to conduct a secretomic analysis. Methods Genome-wide prediction Many secreted proteins have an N-terminal peptide sequence that signals for the translated protein to move into the endoplasmic reticulum where the processing occurs that will ultimately lead to secretion. The presence of these signal peptides can be used to predict the secretome of a cell. Some secretory proteins do not have classical signal peptide sequences. These are called 'leaderless secretory proteins' (LSPs). Genome-wide prediction methods have a variety of problems. There is a high possibility of false positives and false negatives. In addition, gene expression is heavily influenced by environmental conditions, meaning a secretome predicted from the genome or a cDNA library is not likely to match completely with the true secretome. Proteomic approaches are necessary to validate any predicted secreted proteins. Several genome-wide secretome databases or knowledgebases are available based on both curation and computational prediction. These databases include the fungal secretome database (FSD), the fungal secretome knowledgebase (FunSecKB), and the lactic acid bacterial secretome database. The human and animal protein subcellular location database (MetaSecKB) and the protist subcellular proteome database (ProtSecKB) are also recently released. Though there are some inaccuracies in the computational prediction, these databases provide useful resources for further characterizing the protein subcellular locations. Proteomic approaches Mass spectrometry analysis is integral to secretomics. Serum or supernatant containing secreted proteins is digested with a protease and the proteins are separated by 2D gel electrophoresis or chromatographic methods. Each individual