Proteomics is the study of the protein in an organism. Protein is a fundamental building block of life, and proteins are the workhorses within and between cells. Biochemical pathways are built out of enzymes and ligands—without them nothing would be accomplished; plants wouldn’t produce glucose, animals wouldn’t be able to digest food, the immune system would cease to exist, and all other biological processes would grind to a halt. The fundamental importance of proteins for life makes them an important topic of study. The first step in understanding protein structure and function is to extract them. Protein extraction is the process of isolating and purifying protein from samples of whole tissue, cell cultures, or biological fluids. The protein extraction protocol used is tailored to match the starting material and the end goals of the assay. Considering the goal of the experiment is extremely important when developing a protein extraction protocol because certain buffer choices (such as high salt, high detergent formulations) can ruin an experiment when higher order protein structure and function needs to be preserved.
Cell lysis is the first step of breaking the cell membrane that enables further study of the proteins, nucleic acids, and other molecules inside of cells. When cell lysis is successful, the undamaged contents of the cell escape through the damaged cell membrane. These contents are then separated out of the mixed sample and used for further study. The methods used for separation of the lysed cell contents are dependent on the goal of the study. Careful investigation of these inner workings can reveal disease patterns, improve our understanding of normal cellular function, and elucidate biochemical pathways and therapeutic targets. Protein isolation is different from nucleic acid separation, and the reagents used vary drastically. There are a few ways to lyse the cell membrane; these include mechanical disruption, liquid homogenization, freeze/thaw cycles, manual griding, and the use of detergents. Sonication cell lysis is an example of mechanical disruption used for releasing the contents of cells.
Flow cytometry, or fluorescent activated cell sorting (FACS) has become a fundamental method for analyzing and collecting cell populations. Flow cytometry tells you the percentage of cells in a particular population that have the characteristics that you are interested in. These characteristics are defined by the array of surface proteins on each cell. The principle of flow cytometry involves labeling cell surface proteins with fluorophores and using lasers to record the fluorescent profile of a population of cells; these cells can also be sorted and isolated into enriched populations during the FACS process. The results of flow cytometry are read by the technicians and scientists performing the assay, and are typically displayed as two-dimensional dot plots with color density information included for greater detail and dynamic range.
A lateral flow immunoassay is an easy-to-use and inexpensive paper-based device used to detect the presence of specific protein in fluid. The basic immunoassay works by taking advantage of the lock-and-key specificity of antibodies and their corresponding antigens. In the case of a lateral flow immunoassay the capture antibodies are printed onto a paper strip and the liquid moves across it via capillary action. The presence of the target antigen is detected by a colorimetric change on the strip of paper, which also makes the lateral flow assay an example of immunochromatography. The principle component of most immunochromatography devices is usually gold nanoparticles or an enzyme-conjugated bead; the gold nanoparticles have a red hue, and enzyme conjugated beads produce a colorful product when a substrate is introduced into the system. In both instances a positive test result is visible to the naked eye. Most lateral flow immunoassays are qualitative tests, which means that a color change on the test line indicates a positive result while the lack of color indicates a negative result. There is a significant amount of research invested in the development of quantitative lateral flow immunoassays in which numerical analysis of protein concentration is possible.
An antigen is a substance that is capable of stimulating an immune response and activating white blood cells to produce antibodies. Antigens can be proteins or sugars that are located on the outer surfaces of cells. All cells have antigens including the ones inside the body, bacteria, and even viruses. The antibodies produced by the immune system are custom-fitted to the antigen that initially stimulated the immune response. The antibodies have an antigen recognition site (paratope) that has highly specific affinity for a region on the antigen called the epitope.
Nanobeads have applications ranging from basic science research to clinical imaging and targeted drug delivery. Nanobeads are composites of nanoparticles. Nanoparticles are defined as being less than 100 nanometers in diameter while nanobeads are usually around 50 to 200 nanometers in diameter. There are also microbeads, but these are much larger and have diameters of at least 1000 nanometers, or 1 micrometer, which is close to the size of a cell. Animal cells range from 10 to 30 micrometers in diameter. The size of nanobeads is very important to their function; partly because they are so much smaller than a cell, which enables them to be used for cell labeling and isolation. In the case of magnetic nanobeads, the nanometer size imparts the paramagnetic property that is so valuable for biomagnetic separation, clinical imaging (contrast enhanced magnetic resonance (MRI)), and therapeutics such as magnetic hyperthermia for targeted tumor destruction.
As we have entered an age of personalized medicine, we have begun to understand that individual differences play a large role in disease expression and treatment options. This idea is also true of individual cells, the basic unit of life. Therefore, when studying disease phenotypes and developing new drugs, it is becoming increasingly important to study single cells instead of groups of cells. The technologies used for single cell isolation have started to improve and it is now possible to isolate single cells for analysis. Oftentimes, this analysis is referred to as cell omics because it covers a wide range of topics including genomics (DNA), transcriptomics (coding and non-coding RNA), proteomics (protein), and metabolomics (the complete set of small-molecule chemicals found within a biological sample). This is a lot of information to gather from such a small, but powerful structure, and it can become very valuable when studying diseases such as cancer, which generally arises from a mutation within a single cell.
Circulating Tumor Cells (CTCs) are cancerous cells that dissociate from a tumor and circulate throughout the bloodstream. Therefore, the detection of CTCs in the bloodstream is an indicator of cancer progression and an early sign of metastasis. CTCs are not hematopoetic in origin, and they do not express the cell surface marker CD45. However, they do express the surface antigen EpCAM, which is commonly expressed on epithelial cells. Immunomagnetic separation methods take advantage of these surface markers to isolate CTCs from centrifuged blood samples.
Regulation of the pharmaceutical development process is important to ensure that drug products are consistently safe and effective. There are written guidelines for pharmaceutical validation, which ensure that drug compounds are handled and tested properly. There are also separate guidelines for the bioanalytical methods used in the development and testing of new drug compounds. The goal is to standardize and improve the consistency of pharmaceutical studies and that data that are used for drug approval. Examples of analytical methods include ligand binding assays and chromatographic methods (liquid chromatography, gas chromatography, and mass spectrometry). Both the FDA (U.S. Food and Drug Administration) and EMA (European Medicines Agency) regularly update their bioanalytical method validation guidelines, but their focuses are slightly different. The FDA outlines reporting guidelines in more detail, while the EMA focuses more closely on the conduction of experiments. The validation guidelines are unified under ICH (International Council for Harmonisation of Technical requirements for pharmaceuticals for human use).
Magnetic bead suspensions will eventually sediment and aggregate over time. If non-homogeneous biomagnetic separation conditions are used, studies show that the likelihood of irreversible aggregation occurring is very high. Unfortunately, when this happens, the consistency, quality and functionality of the lot are all compromised. There are several steps during the preparation of magnetic beads for diagnostic kits where irreversible aggregation can become a problem unless resuspension techniques are used that guarantee gentle disaggregation.