The proteins secreted by the cells of an organism are collectively referred to as the secretome. Representing up to 30% of the proteome, they include antibodies, cytokines, chemokines, hormones, growth factors, and a vast multitude of other proteins, many of which are involved in essential biological processes. Methods available to study the secretome include DNA microarrays and RNA-Seq, both of which require that the sequence of the target protein be known, and approaches such as mass spectrometry (MS) and microarray technologies, which do not depend on the protein of interest having been sequenced. This article describes some of the complexities of secretome analysis and explains why, among the range of technologies on offer, both antibody microarrays and protein microarrays are increasingly being used for secretome research.
Approaches to secretome analysis vary according to sample type
Samples used for secretome analysis can vary significantly in terms of composition and complexity. “Most secretome studies are performed in vitro on cultured cells,” reports Ilenia Bertipaglia, technical support representative at Grace Bio-Labs. “But while bacteria, yeast, and fungi-derived secretome samples are relatively simple in composition, mammalian cells can be more challenging to work with because they typically require serum or protein additives in their culture media. Serum includes highly abundant proteins such as albumin and IgG that can obscure the identification of much lower abundance secretome proteins, meaning that researchers studying mammalian secretomes need to switch cultured cells to serum-free media for 12–14 hours before collecting conditioned media containing the secreted proteins for analysis.”
Serum proteins can also complicate in vivo secretome studies due to their presence in blood—one of the most widely used sample types owing to its ease of collection and the fact that it contains secreted markers of many organs. “A further problem with analyzing samples such as blood, serum, or plasma to study a particular organ or tissue type is that you can’t necessarily identify which organ the secreted markers originated from,” notes Valerie Jones, Ph.D., director of marketing and technical support at RayBiotech. “If you wish to examine organ-specific secretomes, you need to delve into other peripheral fluids, some of which (urine, saliva, sputum) are easier to collect than others (CSF, aqueous humor). You may also need to study cellular secretomes (i.e. in vitro analysis), or to use extracts prepared from biopsies or organoids to better understand the secretome at an organ or tissue level.”
Studying the secretome presents unique challenges
One of the major challenges of analyzing the secretome concerns the extraordinary dynamic range of the targets of interest, which can span almost 12 orders of magnitude. Experimental complexity is compounded by the sheer number of different proteins present in each sample type, including not only the protein targets of interest, but also intracellular proteins derived from cellular stress and cell death. Accurate detection requires a specific and highly sensitive approach, as exemplified by both antibody microarray and protein microarray technology.
Antibody microarrays benefit from a straightforward workflow
“Antibody microarray technologies have been widely cited as useful tools to survey the secretome,” says Jones. “One reason for this is the nature of the workflow, which simply involves applying the sample to the array surface and capturing the signal intensity at each spot. A further advantage of using antibody microarrays is that low abundance proteins can be efficiently detected because the technique of solid-phase immunocapture eliminates the need for removal of high abundance proteins (which is required prior to analysis by MS). Although panel size is constrained by the number of available antibodies, array manufacturers are gradually pushing this limit through both production and validation of new antibody reagents. Currently, over 3000 human proteins can be detected by RayBiotech arrays, with a typical experiment producing data in just 1–2 days.”
Protein microarrays provide novel insight
Similarly to antibody microarrays, protein microarray technology is ideally suited to study complex matrices like serum or conditioned media because time-consuming pre-fractionation procedures are not required. “The high sensitivity of protein microarrays allows for the detection of low abundance target proteins, especially when a high protein binding capacity substrate such as ONCYTE® porous nitrocellulose is used for microarray deposition,” notes Bertipaglia. “Moreover, both antibody and protein microarrays are highly customizable and can be readily adapted to analyze a particular subset of biomarkers based on the importance of those targets to defined physiological processes.”
Christian M. Loch, chief executive and science officer at AVMBioMed, explains that reliably monitoring and quantitating the secretome by protein microarray often requires additional in-house capabilities for protein separation, enrichment, and scale-up, to complement the fundamental proteomic technology. In AVMBioMed’s case, the company’s proteomic technology uses microarrays containing approximately 20,000 different human proteins to reveal valuable information about complex samples such as cells or serum. By allowing the sample to alter the bound proteins at the same time that it manipulates equivalent proteins endogenously, researchers can investigate the active proteomic state of the sample in a near-physiological context.
“Some secreted proteins are especially difficult to work with,” reports Loch. “For example, certain protein families are of such low abundance that extremely large in vitro cell cultures and multiple extra handling steps (such as those described above) are necessary to obtain usable amounts.” He highlights as an example a recent project requiring that 10 L cells be grown to obtain enough spent media for detectable amounts of the secreted proteins of interest—a family of specifically modified proteins that will be used as biomarkers for early detection of disease. “A further complication of this protein family was that it was very poorly characterized owing both to PTM and to various biochemical and proteomic challenges like resistance to cleavage by proteases making the individual proteins virtually invisible to standard MS,” he says. “By scaling up the proteins, separating them by 2DGE, excising them, and then carrying out further processing to make them amenable to identification, we were able to provide our sister company, Rockland Immunochemicals, with all the information necessary for manufacture of sensitive and highly specific antibodies that will be used in clinical assays.”
Importance to drug discovery
Secretome studies are increasingly being used to advance researchers’ understanding of disease pathology, support personalized treatment, and drive drug discovery efforts. “Researchers are already accustomed to using cell culture-based screens to characterize a drug, and subsequent in vivo experiments to monitor side effects,” summarizes Jones. “By combining these studies with secretome analysis, it is possible to identify other important drug properties. For instance, using our antibody microarrays, researchers showed HGF secretion to be a mechanism of cancer drug resistance. This discovery led to the identification of a combination of drugs that could overcome this resistance through blockade of HGF.”