The secretome is involved in almost all physiological, developmental, and pathological processes. Comprising up to 30% of the proteome, it includes many different types of secreted protein. Researchers typically study sub-sets of secretome proteins to discover how they function in conditions of both health and disease. But while secreted proteins can be readily detected by most immunoassay techniques, a major challenge of secretome analysis lies in the high dynamic range of protein abundance and the sheer numbers of proteins involved.
Because antibody arrays enable highly specific detection of both low and high abundance proteins within the same sample, they are widely used to study the secretome. Moreover, since the panel of analytes can be pre-selected based on established importance in key physiological processes, antibody arrays offer deeper insight than many other techniques used for secretome analysis. In this ebook, we delve into the role of the secretome and look at how using antibody arrays may shed light on its involvement in conditions including COVID-19 and cancer.
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Download this ebook now to learn more about the function and importance of the secretome as well as how to overcome the complexities of secretome analysis.What is the secretome?
The term secretome first came into use in 2004. It was coined by Tjalsma et al who, while investigating the role of secreted proteins in supporting bacterial survival, used it as a collective term to describe all of the secreted proteins and the secretory machinery of a cell. In 2010, the definition was revised to reflect the incredible fluidity of secretome composition and turnover.
The secretome is now recognized as “the global group of secreted proteins into the extracellular space by a cell, tissue, organ, or organism at any given time and conditions through known and unknown secretory mechanisms involving constitutive and regulated secretory organelles.” Various other terms have since become widely used within the field of secretomics, including the matrisome, a core set of extracellular matrix (ECM) proteins, plus proteins known to associate with or modify the ECM; the adhesome, a group of proteins involved in cell-cell and cell-matrix adhesion; and the receptome, namely cellular receptors, many of which are specifically targeted by secreted proteins.
What is the function of the secretome?
Comprising up to 30% of an organism’s proteome, the secretome is essential to almost all physiological, developmental, and pathological processes. These encompass intracellular and intercellular signaling, cell migration, and proliferation, as well as innate and adaptive immunity, wound healing, and digestion, to name but a few. Secretome proteins include antibodies, cytokines, chemokines, hormones, and ECM proteins, as well as growth factors, coagulation factors, adhesion molecules, proteases, and kinases. A vast assortment of other secretome proteins has also been identified, many of which perform critical biological functions.
What is the secretory pathway?
Proteins that are destined to be secreted from mammalian cells usually follow the secretory pathway. After synthesis, a signal peptide consisting of a short, hydrophobic N-terminal sequence targets such proteins to the endoplasmic reticulum (ER), from where they are transported to the Golgi apparatus. Here, they are packed into secretory vesicles, which then move to the plasma membrane. When the secretory vesicles and the plasma membrane merge, the protein contents are released from the cell by exocytosis. As proteins progress through the secretory pathway, they undergo modifications such as glycosylation, specific proteolytic cleavages, disulfide bond formation, protein folding, and assembly into multimers, allowing them to carry out their specified function.
How do researchers study the secretome?
Unlike proteins that are anchored to the cell membrane, which require techniques such as flow cytometry or IHC for detection, and in contrast to cytoplasmic proteins that necessitate cells being lyzed, secreted proteins are relatively straightforward to detect. Commonly used methods include immunoassay techniques such as single-analyte ELISA, antibody arrays (multiplex ELISA), and bead-based arrays, as well as mass spectrometry.
The complexity of secretome analysis increases according to the number of targets requiring detection and the nature of the sample material. While blood, serum, and plasma are most commonly used, other sample types include peripheral fluids such as urine, saliva, and sputum, as well as cerebrospinal fluid (CSF), aqueous humor, and tumor interstitial fluid that are more difficult to collect. Secretome analysis is also performed in vitro, using cultured cells, organoids, or tissue biopsy fluid, whereas investigating bacterial or fungal secretomes allows researchers to conduct pathogenic or microbiome-related studies.
What are the advantages of using antibody arrays for secretome analysis?
Antibody array technologies have been widely cited as useful tools to study the secretome, mainly due to the combination of sensitivity and specificity afforded by antibody use. A typical workflow involves simply applying the sample to the array surface and measuring the signal intensity at each spot, with an experiment generating data in just one or two days. Because the technique of solid-phase immunocapture obviates the need for removal of high abundance proteins (which is required prior to analysis by mass spectrometry), antibody arrays allow for highly efficient detection of low abundance targets. Moreover, the panel of analytes can be pre-selected based on established importance in key physiological processes, allowing for highly relevant discoveries. Although the size of antibody array panels is restricted to the number of available antibodies, array manufacturers are gradually pushing this limit by producing and validating new antibody reagents. Currently, over 3,000 human proteins can be detected using RayBiotech’s antibody arrays.
How are antibody arrays being used to study the secretome?
Antibody arrays are employed in many different guises for secretome analysis. Comparing normal sample material with material pertaining to a particular disease state enables identification of aberrant protein secretions that could indicate a pathologic condition, whereas observing the secretome profile in cell culture with and without a drug allows for identification of important drug properties. Recently, RayBiotech’s antibody arrays have highlighted the potential for mesenchymal stem cell (MSC) secretome-based therapies to treat joint disease, and have also been used to identify secreted factors in tumor stromal cells that contribute to cancer drug resistance. These are just two examples of the many applications of antibody array technology within secretome research.
To learn more about the secretome, download our free eBook now.