Secretomics
Encyclopedia
Secretomics is a subset of proteomics
in which all of the secreted proteins
of a cell, tissue, or organism are analyzed. Secreted proteins are involved in a variety of physiological processes, including cell signaling
and matrix
remodeling, but are also integral to invasion and metastasis
of malignant cells. Secretomics has thus been especially important in the discovery of biomarkers for cancer
.
. They defined the secretome as all of the secreted proteins and secretory machinery of the bacteria. Using a database of protein sequences in B. subtilis and an algorithm that looked at cleavage sites and amino-terminal signal peptides
characteristic of secreted proteins they were able to predict what fraction of the proteome is secreted by the cell. In 2001 the same lab set a standard of secretomics – predictions based on amino acid sequence alone are not enough to define the secretome. They used two-dimensional gel electrophoresis
and mass spectrometry
to identify 82 proteins secreted by B. subtilis, only 48 of which had been predicted using the genome
-based method of their previous paper. This demonstrates the need for protein verification of predicted findings.
As the complicated nature of secretory pathways was revealed – namely that there are many non-classical pathways of secretion and there are many non-secreted proteins that are a part of the classical secretory pathway – a more in-depth definition of the secretome became necessary. In 2010, Agrawal et al. suggested defining the secretome 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.”
from cell culture media and cellular debris can contaminate the collection of secreted proteins used for analysis. Bovine contaminants present a particular challenge because the protein sequences of many bovine extracellular proteins, like fibronectin
and fibulin-1
, are similar to the human protein sequences. To remove these contaminants, cells can be washed with PBS
or serum-free medium
(SFM) before incubating in SFM and collecting secreted proteins. Care must be taken not 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.
that can detect even single molecules of a protein.
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.
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. Software like SignalP can identify signal sequences (and their cleavage sites) to predict proteins that are secreted. Since transmembrane proteins
are also processed in the ER, but not secreted, software like the TMHMM server is used to predict transmembrane domains and therefore eliminate false positives. Some secretory proteins do not have classical signal peptide sequences. These ‘leaderless secretory proteins’ (LSPs) will be missed by SignalP. SecretomeP is a software that has been developed to try to predict these non-classical secretory proteins from their sequences. Genome-wide secretomes have been predicted for a wide range of organisms, including human, mouse, zebrafish, and hundreds of bacteria.
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.
and the proteins are separated by 2D gel electrophoresis or chromatographic
methods. Each individual protein is then analyzed by mass spectrometry and the peptide-mass fingerprint
generated can be run through a database to identify the protein.
Stable isotope labeling by amino acids in cell culture (SILAC) has emerged as an important method in secretomics – it helps to distinguish between secreted proteins and bovine serum contaminants in cell culture. Supernatant from cells grown in normal medium and cells grown in medium with stable-isotope labeled amino acids is mixed in a 1:1 ratio and analyzed by mass spectrometry. Protein contaminants in the serum will only show one peak because they do not have a labeled equivalent. As an example, the SILAC method has been used successfully to distinguish between proteins secreted by human chondrocytes
in culture and serum contaminants.
An antibody microarray is a highly sensitive and high-throughput method for protein detection that has recently become part of secretomic analysis. Antibodies
, or another type of binder molecule, are fixed onto a solid support and a fluorescently labeled protein mixture is added. Signal intensities are used to identify proteins. Antibody microarrays are extremely versatile – they can be used to analyze the amount of protein in a mixture, different protein isoforms
, posttranslational modifications
, and the biochemical activity of proteins. In addition, these microarrays are highly sensitive – they can detect single molecules of protein. Antibody microarrays are currently being used mostly to analyze human plasma
samples but can also be used for cultured cells and body fluid secretomics, presenting a simple way to look for the presence of many proteins at one time.
through cell growth, migration, invasion, and angiogenesis
, making secretomics an excellent method for the discovery of cancer biomarkers. Using a body fluid or full serum proteomic method to identify biomarkers can be extremely difficult – body fluids are complex and highly variable. Secretomic analysis of cancer cell lines or diseased tissue presents a simpler and more specific alternative for biomarker discovery.
The two main biological sources for cancer secretomics are cancer cell line supernatants and proximal biological fluids, the fluids in contact with a tumor
. Cancer cell line supernatant is an attractive source of secreted proteins. There are many standardized cell lines available and supernatant is much simpler to analyze than proximal body fluid. But it is unclear whether a cell line secretome is a good representation of an actual tumor in its specific microenvironment and a standardized cell line is not illustrative of the heterogeneity of a real tumor. Analysis of proximal fluids can give a better idea of a human tumor secretome, but this method also has its drawbacks. Procedures for collecting proximal fluids still need to be standardized and non-malignant controls are needed. In addition, environmental and genetic differences between patients can complicate analysis.
Secretomic analysis has discovered potential new biomarkers in many cancer types, including lung cancer
, liver cancer
, pancreatic cancer
, colorectal cancer
, prostate cancer
, and breast cancer
. Prostate-specific antigen (PSA), the current standard biomarker for prostate cancer, has a low diagnostic specificity – PSA levels can not always discriminate between aggressive and non-aggressive cancer – and so a better biomarker is greatly needed. Using secretomic analysis of prostate cell lines, one study was able to discover multiple proteins found in higher levels in the serum of cancer patients than in healthy controls.
There is also a great need for biomarkers for the detection of breast cancer – currently biomarkers only exist for monitoring later stages of cancer. Secretomic analysis of breast cancer cell lines led to the discovery of the protein ALCAM
as a new biomarker with promising diagnostic potential.
. In IVF
, embryos are assessed on morphological criteria in an attempt to find those with high implantation potential. Finding a more quantitative method of assessment could help reduce the number of embryos used in IVF, thereby reducing higher order pregnancies
. For example, one study was able to develop secretome fingerprints for many blastocysts
and found 9 proteins that could distinguish between blastocysts with normal and abnormal numbers of chromosomes
. This type of analysis could help replace preimplantation genetic screening
(PGS), which involves biopsy of embryonic cells and can be harmful to development.
Proteomics
Proteomics is the large-scale study of proteins, particularly their structures and functions. Proteins are vital parts of living organisms, as they are the main components of the physiological metabolic pathways of cells. The term "proteomics" was first coined in 1997 to make an analogy with...
in which all of the secreted proteins
Secretory protein
A secretory protein is any protein, whether it be endocrine or exocrine, which is secreted by a cell. Secretory proteins include many hormones, enzymes, toxins, and antimicrobial peptides.Secretory proteins are synthesized in endoplasmic reticulum....
of a cell, tissue, or organism are analyzed. Secreted proteins are involved in a variety of physiological processes, including cell signaling
Cell signaling
Cell signaling is part of a complex system of communication that governs basic cellular activities and coordinates cell actions. The ability of cells to perceive and correctly respond to their microenvironment is the basis of development, tissue repair, and immunity as well as normal tissue...
and matrix
Extracellular matrix
In biology, the extracellular matrix is the extracellular part of animal tissue that usually provides structural support to the animal cells in addition to performing various other important functions. The extracellular matrix is the defining feature of connective tissue in animals.Extracellular...
remodeling, but are also integral to invasion and metastasis
Metastasis
Metastasis, or metastatic disease , is the spread of a disease from one organ or part to another non-adjacent organ or part. It was previously thought that only malignant tumor cells and infections have the capacity to metastasize; however, this is being reconsidered due to new research...
of malignant cells. Secretomics has thus been especially important in the discovery of biomarkers for cancer
Cancer
Cancer , known medically as a malignant neoplasm, is a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the...
.
History of the secretome
In 2000 Tjalsma et al. coined the term ‘secretome’ in their study of the eubacterium B. subtilisBacillus subtilis
Bacillus subtilis, known also as the hay bacillus or grass bacillus, is a Gram-positive, catalase-positive bacterium commonly found in soil. A member of the genus Bacillus, B. subtilis is rod-shaped, and has the ability to form a tough, protective endospore, allowing the organism to tolerate...
. They defined the secretome as all of the secreted proteins and secretory machinery of the bacteria. Using a database of protein sequences in B. subtilis and an algorithm that looked at cleavage sites and amino-terminal signal peptides
Signal peptide
A signal peptide is a short peptide chain that directs the transport of a protein.Signal peptides may also be called targeting signals, signal sequences, transit peptides, or localization signals....
characteristic of secreted proteins they were able to predict what fraction of the proteome is secreted by the cell. In 2001 the same lab set a standard of secretomics – predictions based on amino acid sequence alone are not enough to define the secretome. They used two-dimensional gel electrophoresis
Two-dimensional gel electrophoresis
Two-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins...
and mass spectrometry
Mass spectrometry
Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of charged particles.It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and...
to identify 82 proteins secreted by B. subtilis, only 48 of which had been predicted using the genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
-based method of their previous paper. This demonstrates the need for protein verification of predicted findings.
As the complicated nature of secretory pathways was revealed – namely that there are many non-classical pathways of secretion and there are many non-secreted proteins that are a part of the classical secretory pathway – a more in-depth definition of the secretome became necessary. In 2010, Agrawal et al. suggested defining the secretome 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.”
Contaminants
In culture, cells are surrounded by contaminants. Bovine serumFetal bovine serum
Fetal bovine serum or FBS is the portion of plasma remaining after coagulation of blood, during which process the plasma protein fibrinogen is converted to fibrin and remains behind in the clot. Fetal bovine serum comes from the blood drawn from a bovine fetus via a closed system of collection at...
from cell culture media and cellular debris can contaminate the collection of secreted proteins used for analysis. Bovine contaminants present a particular challenge because the protein sequences of many bovine extracellular proteins, like fibronectin
Fibronectin
Fibronectin is a high-molecular weight glycoprotein of the extracellular matrix that binds to membrane-spanning receptor proteins called integrins. In addition to integrins, fibronectin also binds extracellular matrix components such as collagen, fibrin and heparan sulfate proteoglycans...
and fibulin-1
FBLN1
FBLN1 is the gene encoding fibulin-1, an extracellular matrix and plasma protein.- Function :Fibulin-1 is a secreted glycoprotein that is found in association with extracellular matrix structures including fibronectin-containing fibers, elastin-containing fibers and basement membranes...
, are similar to the human protein sequences. To remove these contaminants, cells can be washed with PBS
Phosphate buffered saline
Phosphate buffered saline is a buffer solution commonly used in biological research. It is a water-based salt solution containing sodium chloride, sodium phosphate, and, in some formulations, potassium chloride and potassium phosphate. The buffer's phosphate groups help to maintain a constant pH...
or serum-free medium
Growth medium
A growth medium or culture medium is a liquid or gel designed to support the growth of microorganisms or cells, or small plants like the moss Physcomitrella patens.There are different types of media for growing different types of cells....
(SFM) before incubating in SFM and collecting secreted proteins. Care must be taken not 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 microarraysAntibody microarray
An antibody microarray is a specific form of protein microarrays, a collection of capture antibodies are spotted and fixed on a solid surface, such as glass, plastic and silicon chip for the purpose of detecting antigens...
that can detect even single molecules of a protein.
Relevance of in vitro studies
Many secretomic studies are conducted in vitroIn vitro
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
with cell culture methods, but it is unclear whether the same proteins are secreted in vivo
In vivo
In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
. 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.
Genome-wide prediction
Many secreted proteins have an N-terminal peptide sequence that signals for the translated protein to move into the endoplasmic reticulumEndoplasmic reticulum
The endoplasmic reticulum is an organelle of cells in eukaryotic organisms that forms an interconnected network of tubules, vesicles, and cisternae...
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. Software like SignalP can identify signal sequences (and their cleavage sites) to predict proteins that are secreted. Since transmembrane proteins
Transmembrane protein
A transmembrane protein is a protein that goes from one side of a membrane through to the other side of the membrane. Many TPs function as gateways or "loading docks" to deny or permit the transport of specific substances across the biological membrane, to get into the cell, or out of the cell as...
are also processed in the ER, but not secreted, software like the TMHMM server is used to predict transmembrane domains and therefore eliminate false positives. Some secretory proteins do not have classical signal peptide sequences. These ‘leaderless secretory proteins’ (LSPs) will be missed by SignalP. SecretomeP is a software that has been developed to try to predict these non-classical secretory proteins from their sequences. Genome-wide secretomes have been predicted for a wide range of organisms, including human, mouse, zebrafish, and hundreds of bacteria.
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
Complementary DNA
In genetics, complementary DNA is DNA synthesized from a messenger RNA template in a reaction catalyzed by the enzyme reverse transcriptase and the enzyme DNA polymerase. cDNA is often used to clone eukaryotic genes in prokaryotes...
library is not likely to match completely with the true secretome. Proteomic approaches are necessary to validate any predicted secreted proteins.
Proteomic approaches
Mass spectrometry analysis is integral to secretomics. Serum or supernatant containing secreted proteins is digested with a proteaseProtease
A protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain forming the protein....
and the proteins are separated by 2D gel electrophoresis or chromatographic
Chromatography
Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures....
methods. Each individual protein is then analyzed by mass spectrometry and the peptide-mass fingerprint
Peptide-mass fingerprint
In bio-informatics, a peptide-mass fingerprint or peptide-mass map is a mass spectrum of a mixture of peptides that comes from a digested protein being analyzed. The mass spectrum serves as a fingerprint in the sense that it is a pattern that can serve to identify the protein....
generated can be run through a database to identify the protein.
Stable isotope labeling by amino acids in cell culture (SILAC) has emerged as an important method in secretomics – it helps to distinguish between secreted proteins and bovine serum contaminants in cell culture. Supernatant from cells grown in normal medium and cells grown in medium with stable-isotope labeled amino acids is mixed in a 1:1 ratio and analyzed by mass spectrometry. Protein contaminants in the serum will only show one peak because they do not have a labeled equivalent. As an example, the SILAC method has been used successfully to distinguish between proteins secreted by human chondrocytes
Chondrocyte
Chondrocytes are the only cells found in cartilage. They produce and maintain the cartilaginous matrix, which consists mainly of collagen and proteoglycans...
in culture and serum contaminants.
An antibody microarray is a highly sensitive and high-throughput method for protein detection that has recently become part of secretomic analysis. Antibodies
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
, or another type of binder molecule, are fixed onto a solid support and a fluorescently labeled protein mixture is added. Signal intensities are used to identify proteins. Antibody microarrays are extremely versatile – they can be used to analyze the amount of protein in a mixture, different protein isoforms
Protein isoform
A protein isoform is any of several different forms of the same protein. Different forms of a protein may be produced from related genes, or may arise from the same gene by alternative splicing. A large number of isoforms are caused by single-nucleotide polymorphisms or SNPs, small genetic...
, posttranslational modifications
Posttranslational modification
Posttranslational modification is the chemical modification of a protein after its translation. It is one of the later steps in protein biosynthesis, and thus gene expression, for many proteins....
, and the biochemical activity of proteins. In addition, these microarrays are highly sensitive – they can detect single molecules of protein. Antibody microarrays are currently being used mostly to analyze human plasma
Blood plasma
Blood plasma is the straw-colored liquid component of blood in which the blood cells in whole blood are normally suspended. It makes up about 55% of the total blood volume. It is the intravascular fluid part of extracellular fluid...
samples but can also be used for cultured cells and body fluid secretomics, presenting a simple way to look for the presence of many proteins at one time.
Discovery of cancer biomarkers
Besides being important in normal physiological processes, secreted proteins also have an integral role in tumorigenesisCarcinogenesis
Carcinogenesis or oncogenesis is literally the creation of cancer. It is a process by which normal cells are transformed into cancer cells...
through cell growth, migration, invasion, and angiogenesis
Angiogenesis
Angiogenesis is the physiological process involving the growth of new blood vessels from pre-existing vessels. Though there has been some debate over terminology, vasculogenesis is the term used for spontaneous blood-vessel formation, and intussusception is the term for the formation of new blood...
, making secretomics an excellent method for the discovery of cancer biomarkers. Using a body fluid or full serum proteomic method to identify biomarkers can be extremely difficult – body fluids are complex and highly variable. Secretomic analysis of cancer cell lines or diseased tissue presents a simpler and more specific alternative for biomarker discovery.
The two main biological sources for cancer secretomics are cancer cell line supernatants and proximal biological fluids, the fluids in contact with a tumor
Tumor
A tumor or tumour is commonly used as a synonym for a neoplasm that appears enlarged in size. Tumor is not synonymous with cancer...
. Cancer cell line supernatant is an attractive source of secreted proteins. There are many standardized cell lines available and supernatant is much simpler to analyze than proximal body fluid. But it is unclear whether a cell line secretome is a good representation of an actual tumor in its specific microenvironment and a standardized cell line is not illustrative of the heterogeneity of a real tumor. Analysis of proximal fluids can give a better idea of a human tumor secretome, but this method also has its drawbacks. Procedures for collecting proximal fluids still need to be standardized and non-malignant controls are needed. In addition, environmental and genetic differences between patients can complicate analysis.
Secretomic analysis has discovered potential new biomarkers in many cancer types, including lung cancer
Lung cancer
Lung cancer is a disease characterized by uncontrolled cell growth in tissues of the lung. If left untreated, this growth can spread beyond the lung in a process called metastasis into nearby tissue and, eventually, into other parts of the body. Most cancers that start in lung, known as primary...
, liver cancer
Liver cancer
Liver tumors or hepatic tumors are tumors or growths on or in the liver . Several distinct types of tumors can develop in the liver because the liver is made up of various cell types. These growths can be benign or malignant...
, pancreatic cancer
Pancreatic cancer
Pancreatic cancer refers to a malignant neoplasm of the pancreas. The most common type of pancreatic cancer, accounting for 95% of these tumors is adenocarcinoma, which arises within the exocrine component of the pancreas. A minority arises from the islet cells and is classified as a...
, colorectal cancer
Colorectal cancer
Colorectal cancer, commonly known as bowel cancer, is a cancer caused by uncontrolled cell growth , in the colon, rectum, or vermiform appendix. Colorectal cancer is clinically distinct from anal cancer, which affects the anus....
, prostate cancer
Prostate cancer
Prostate cancer is a form of cancer that develops in the prostate, a gland in the male reproductive system. Most prostate cancers are slow growing; however, there are cases of aggressive prostate cancers. The cancer cells may metastasize from the prostate to other parts of the body, particularly...
, and breast cancer
Breast cancer
Breast cancer is cancer originating from breast tissue, most commonly from the inner lining of milk ducts or the lobules that supply the ducts with milk. Cancers originating from ducts are known as ductal carcinomas; those originating from lobules are known as lobular carcinomas...
. Prostate-specific antigen (PSA), the current standard biomarker for prostate cancer, has a low diagnostic specificity – PSA levels can not always discriminate between aggressive and non-aggressive cancer – and so a better biomarker is greatly needed. Using secretomic analysis of prostate cell lines, one study was able to discover multiple proteins found in higher levels in the serum of cancer patients than in healthy controls.
There is also a great need for biomarkers for the detection of breast cancer – currently biomarkers only exist for monitoring later stages of cancer. Secretomic analysis of breast cancer cell lines led to the discovery of the protein ALCAM
ALCAM
CD166 antigen is a protein that in humans is encoded by the ALCAM gene.. It is also called CD166 .-External links:...
as a new biomarker with promising diagnostic potential.
Assisted Reproductive Technologies
Analyzing the human embryonic secretome could be helpful in finding a non-invasive method for determining viability of embryosEmbryo
An embryo is a multicellular diploid eukaryote in its earliest stage of development, from the time of first cell division until birth, hatching, or germination...
. In IVF
In vitro fertilisation
In vitro fertilisation is a process by which egg cells are fertilised by sperm outside the body: in vitro. IVF is a major treatment in infertility when other methods of assisted reproductive technology have failed...
, embryos are assessed on morphological criteria in an attempt to find those with high implantation potential. Finding a more quantitative method of assessment could help reduce the number of embryos used in IVF, thereby reducing higher order pregnancies
Multiple birth
A multiple birth occurs when more than one fetus is carried to term in a single pregnancy. Different names for multiple births are used, depending on the number of offspring. Common multiples are two and three, known as twins and triplets...
. For example, one study was able to develop secretome fingerprints for many blastocysts
Blastocyst
The blastocyst is a structure formed in the early embryogenesis of mammals, after the formation of the morula. It is a specifically mammalian example of a blastula. It possesses an inner cell mass , or embryoblast, which subsequently forms the embryo, and an outer layer of cells, or trophoblast,...
and found 9 proteins that could distinguish between blastocysts with normal and abnormal numbers of chromosomes
Chromosome
A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.Chromosomes...
. This type of analysis could help replace preimplantation genetic screening
Preimplantation genetic diagnosis
In medicine and genetics pre-implantation genetic diagnosis refers to procedures that are performed on embryos prior to implantation, sometimes even on oocytes prior to fertilization. PGD is considered another way to prenatal diagnosis...
(PGS), which involves biopsy of embryonic cells and can be harmful to development.