Secretomics is a type of
proteomics
Proteomics is the large-scale study of proteins. Proteins are vital macromolecules of all living organisms, with many functions such as the formation of structural fibers of muscle tissue, enzymatic digestion of food, or synthesis and replicatio ...
which involves the analysis of the
secretome The secretome is the set of proteins expressed by an organism and secreted into the extracellular space. In humans, this subset of the proteome encompasses 13-20% of all proteins, including cytokines, growth factors, extracellular matrix proteins ...
—all the
secreted proteins of a cell, tissue or organism.
Secreted proteins are involved in a variety of physiological processes, including
cell signaling
In biology, cell signaling (cell signalling in British English) is the Biological process, process by which a Cell (biology), cell interacts with itself, other cells, and the environment. Cell signaling is a fundamental property of all Cell (biol ...
and
matrix
Matrix (: matrices or matrixes) or MATRIX may refer to:
Science and mathematics
* Matrix (mathematics), a rectangular array of numbers, symbols or expressions
* Matrix (logic), part of a formula in prenex normal form
* Matrix (biology), the m ...
remodeling, but are also integral to invasion and
metastasis
Metastasis is a pathogenic agent's spreading from an initial or primary site to a different or secondary site within the host's body; the term is typically used when referring to metastasis by a cancerous tumor. The newly pathological sites, ...
of
malignant cells.
Secretomics has thus been especially important in the discovery of
biomarker
In biomedical contexts, a biomarker, or biological marker, is a measurable indicator of some biological state or condition. Biomarkers are often measured and evaluated using blood, urine, or soft tissues to examine normal biological processes, ...
s for
cancer
Cancer is a group of diseases involving Cell growth#Disorders, abnormal cell growth with the potential to Invasion (cancer), invade or Metastasis, spread to other parts of the body. These contrast with benign tumors, which do not spread. Po ...
and understanding molecular basis of pathogenesis. The analysis of the insoluble fraction of the secretome (the
extracellular matrix
In biology, the extracellular matrix (ECM), also called intercellular matrix (ICM), is a network consisting of extracellular macromolecules and minerals, such as collagen, enzymes, glycoproteins and hydroxyapatite that provide structural and bio ...
) has been termed matrisomics.
History of the secretome
In 2000 Tjalsma et al. coined the term 'secretome' in their study of the eubacterium ''
B. subtilis''. 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 peptide
A signal peptide (sometimes referred to as signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide) is a short peptide (usually 16–30 amino acids long) present at the ...
s 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
Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a ''mass spectrum'', a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is used ...
to identify 82 proteins secreted by ''B. subtilis'', only 48 of which had been predicted using the
genome
A genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding genes, other functional regions of the genome such as ...
-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".
Challenges of secretomic analysis
Contaminants
In culture, cells are surrounded by contaminants.
Bovine serum 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 is a high- molecular weight (~500-~600 kDa) glycoprotein of the extracellular matrix that binds to membrane-spanning receptor proteins called integrins. Fibronectin also binds to other extracellular matrix proteins such as col ...
and
fibulin-1, are similar to the human protein sequences.
[ To remove these contaminants, cells can be washed with ]PBS
The Public Broadcasting Service (PBS) is an American public broadcaster and non-commercial, free-to-air television network based in Arlington, Virginia. PBS is a publicly funded nonprofit organization and the most prominent provider of educat ...
or serum-free medium
Medium may refer to:
Aircraft
*Medium bomber, a class of warplane
* Tecma Medium, a French hang glider design Arts, entertainment, and media Films
* ''The Medium'' (1921 film), a German silent film
* ''The Medium'' (1951 film), a film vers ...
(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 microarray
An antibody microarray (also known as antibody array) is a specific form of protein microarray. In this technology, a collection of captured antibodies are spotted and fixed on a solid surface such as glass, plastic, membrane, or silicon chip, an ...
s that can detect even single molecules of a protein.
Relevance of ''in vitro'' studies
Many secretomic studies are conducted ''in vitro
''In vitro'' (meaning ''in glass'', or ''in the glass'') Research, studies are performed with Cell (biology), cells or biological molecules outside their normal biological context. Colloquially called "test-tube experiments", these studies in ...
'' with cell culture methods, but it is unclear whether the same proteins are secreted ''in vivo
Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, an ...
''. 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
The endoplasmic reticulum (ER) is a part of a transportation system of the eukaryote, eukaryotic cell, and has many other important functions such as protein folding. The word endoplasmic means "within the cytoplasm", and reticulum is Latin for ...
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
In genetics, complementary DNA (cDNA) is DNA that was reverse transcribed (via reverse transcriptase) from an RNA (e.g., messenger RNA or microRNA). cDNA exists in both single-stranded and double-stranded forms and in both natural and engin ...
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
A protease (also called a peptidase, proteinase, or proteolytic enzyme) is an enzyme that catalysis, catalyzes proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the formation of new protein products ...
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
Stable isotope labeling by/with amino acids in cell culture (SILAC) is a technique based on mass spectrometry that detects differences in protein abundance among samples using non-radioactive isotopic labeling. It is a popular method for quantitat ...
(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 ]chondrocyte
Chondrocytes (, ) are the only cells found in healthy cartilage. They produce and maintain the cartilaginous matrix, which consists mainly of collagen and proteoglycans. Although the word '' chondroblast'' is commonly used to describe an immatu ...
s 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
An antibody (Ab) or immunoglobulin (Ig) is a large, Y-shaped protein belonging to the immunoglobulin superfamily which is used by the immune system to identify and neutralize antigens such as bacteria and viruses, including those that caus ...
, 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 isoform
A protein isoform, or "protein variant", is a member of a set of highly similar proteins that originate from a single gene and are the result of genetic differences. While many perform the same or similar biological roles, some isoforms have uniqu ...
s, posttranslational modification
In molecular biology, post-translational modification (PTM) is the covalent process of changing proteins following protein biosynthesis. PTMs may involve enzymes or occur spontaneously. Proteins are created by ribosomes, which translate mRNA ...
s, 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.[
]
Implications and significance
Discovery of cancer biomarkers
Besides being important in normal physiological processes, secreted proteins also have an integral role in tumorigenesis
Carcinogenesis, also called oncogenesis or tumorigenesis, is the formation of a cancer, whereby normal cells are transformed into cancer cells. The process is characterized by changes at the cellular, genetic, and epigenetic levels and abn ...
through cell growth, migration, invasion, and angiogenesis
Angiogenesis is the physiological process through which new blood vessels form from pre-existing vessels, formed in the earlier stage of vasculogenesis. Angiogenesis continues the growth of the vasculature mainly by processes of sprouting and ...
, 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
A neoplasm () is a type of abnormal and excessive growth of tissue. The process that occurs to form or produce a neoplasm is called neoplasia. The growth of a neoplasm is uncoordinated with that of the normal surrounding tissue, and persists ...
. 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, also known as lung carcinoma, is a malignant tumor that begins in the lung. Lung cancer is caused by genetic damage to the DNA of cells in the airways, often caused by cigarette smoking or inhaling damaging chemicals. Damaged ...
, liver cancer
Liver cancer, also known as hepatic cancer, primary hepatic cancer, or primary hepatic malignancy, is cancer that starts in the liver. Liver cancer can be primary in which the cancer starts in the liver, or it can be liver metastasis, or secondar ...
, pancreatic cancer
Pancreatic cancer arises when cell (biology), cells in the pancreas, a glandular organ behind the stomach, begin to multiply out of control and form a Neoplasm, mass. These cancerous cells have the malignant, ability to invade other parts of ...
, colorectal cancer
Colorectal cancer (CRC), also known as bowel cancer, colon cancer, or rectal cancer, is the development of cancer from the Colon (anatomy), colon or rectum (parts of the large intestine). Signs and symptoms may include Lower gastrointestinal ...
, prostate cancer
Prostate cancer is the neoplasm, uncontrolled growth of cells in the prostate, a gland in the male reproductive system below the bladder. Abnormal growth of the prostate tissue is usually detected through Screening (medicine), screening tests, ...
, and breast cancer
Breast cancer is a cancer that develops from breast tissue. Signs of breast cancer may include a Breast lump, lump in the breast, a change in breast shape, dimpling of the skin, Milk-rejection sign, milk rejection, fluid coming from the nipp ...
. 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.][
]
Assisted reproductive technologies
Analyzing the human embryonic secretome could be helpful in finding a non-invasive method for determining viability of embryo
An embryo ( ) is the initial stage of development for a multicellular organism. In organisms that reproduce sexually, embryonic development is the part of the life cycle that begins just after fertilization of the female egg cell by the male sp ...
s. 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 blastocyst
The blastocyst is a structure formed in the early embryonic development of mammals. It possesses an inner cell mass (ICM) also known as the ''embryoblast'' which subsequently forms the embryo, and an outer layer of trophoblast cells called the ...
s and found 9 proteins that could distinguish between blastocysts with normal and abnormal numbers of chromosome
A chromosome is a package of DNA containing part or all of the genetic material of an organism. In most chromosomes, the very long thin DNA fibers are coated with nucleosome-forming packaging proteins; in eukaryotic cells, the most import ...
s. This type of analysis could help replace preimplantation genetic screening (PGS), which involves biopsy of embryonic cells and can be harmful to development.
References
{{Reflist, 25em
Proteomics
Secretion