Name: proteomics and protein expression
Uploaded: 2023-04-30T11:59:26.000Z
Duration: 1 min 33 s
Channel: JoVE

Proteomics may be defined as the large-scale systematic study of the proteome - the complete set of proteins expressed by an organelle, cell, tissue, or organism at a certain time.
Depending on the study type, either the top-down proteomics approach that analyzes intact proteins from biological systems, or the bottom-up approach that studies the proteins by analyzing the peptide fragments, could be used to obtain information on the target protein set.
In a typical workflow, protein samples are collected, for example, from COVID-19 patients and healthy controls, and digested into peptides. Then,&#160; high-throughput techniques like MS, allow identification and comparative screening of significantly regulated or altered proteins between the study populations, which&#160; can be identified using available databases. This in-depth proteome profiling allows detection of disease-specific protein biomarkers and identification of potential therapeutic targets.
Expression proteomics studies the differences in protein expression between samples. Structural proteomics helps map out the protein structure within a specific organelle, while functional proteomics identifies the biological functions of an

proteomics

Proteomics

A proteome is the entire set of proteins that a cell type produces. We can study proteomes using the knowledge of genomes because genes code for mRNAs, and the mRNAs encode proteins. Although mRNA analysis is a step in the right direction, not all mRNAs are translated into proteins.
Proteomics is the study of proteomes&#39; function. It involves the large-scale systematic study of the proteome to denote the protein complement expressed by a genome. Scientist Mark Wilkins coined the term proteomics describing it as the &#8216;PROTein complement expressed by a genOME.&#8217;
As proteomics often complements genomics, it is useful when scientists want to test their research hypotheses. Even though all the cells in a multicellular organism have the same set of genes, the set of proteins produced in different tissues is different because it is dependent on gene expression. Thus, the genome is constant, but the proteome varies and is dynamic within an organism. In addition, RNAs can be alternately spliced (cut and pasted to create novel combinations and novel proteins). Also, many proteins modify themselves after translation by processes such as proteolytic cleavage, phosphorylation, glycosylation, and ubiquitination. There are also protein-protein interactions, which complicate studying proteomes. Although the genome provides a blueprint, the final architecture depends on several factors that can change the progression of events that generate the proteome. Because of this, there are different types of proteomics, such as&#160; expression, structural and functional, which help study various aspects of the proteins.
The ultimate goal of proteomics is to identify or compare the proteins expressed from a given genome under specific conditions, study the interactions between the proteins, and use the information to predict cell behavior or develop drug targets. Just as analyzing the genome requires basic DNA sequencing technique, proteomics requires techniques for protein analysis. The basic technique for protein analysis is mass spectrometry that identifies and determines a molecule&#39;s characteristics. Advances in spectrometry have allowed researchers to analyze very small protein samples. X-ray crystallography, for example, enables scientists to determine a protein crystal&#39;s three-dimensional structure at atomic resolution. Nuclear magnetic resonance uses atoms&#39; magnetic properties to determine the protein&#39;s three-dimensional structure in an aqueous solution. Scientists have also used protein microarrays to study protein interactions. Large-scale adaptations of the basic two-hybrid screen have provided the basis for protein microarrays. Scientists use computer software to analyze the vast amount of data for proteomic analysis.

A proteome is the entire set of proteins that a cell type produces. We can study proteomes using the knowledge of genomes because genes code for mRNAs, ...

Education

JoVE Core

Cell Biology

Unit 1

Analyzing Cells and Proteins

KEY TERMS AND CONCEPTS

proteomics and protein expression

Proteomics studies the proteins made by a cell type and how those proteins function. A proteome is the full set of proteins produced by a cell type, and proteomics is the large-scale study of that protein set. The term was coined by Mark Wilkins, who described it as the “PROTein complement expressed by a genOME.”

Proteomics is closely linked to genomics because genes lead to mRNAs, and mRNAs usually encode proteins. However, not every mRNA is translated into a protein. This means mRNA data can help, but it does not show the full protein picture.

The proteome is dynamic, while the genome stays constant in a multicellular organism. Cells in different tissues can produce different proteins because gene expression differs from tissue to tissue. RNA can also be alternately spliced, which means it is cut and pasted in new ways to make novel combinations and new proteins.

Proteins can change after translation in several ways. These changes include proteolytic cleavage, phosphorylation, glycosylation, and ubiquitination. Protein-protein interactions also make proteomes harder to study. Even though the genome gives the blueprint, many factors shape the final protein output.

Because of this complexity, scientists use expression, structural, and functional proteomics to study proteins in different ways. The main goals are to identify or compare proteins made from a given genome under specific conditions, study how proteins interact, and use that information to predict cell behavior or find drug targets. Proteomics often complements genomics when researchers want to test a hypothesis.

Protein analysis uses techniques similar in importance to DNA sequencing in genomics. The basic method is mass spectrometry, which identifies a molecule and its characteristics, and newer advances allow analysis of very small protein samples. Scientists also use X-ray crystallography to find a protein crystal’s three-dimensional structure at atomic resolution, and nuclear magnetic resonance to determine structure in an aqueous solution. Protein microarrays help study protein interactions, often by using large-scale versions of the two-hybrid screen, and computer software is needed to manage the large amount of data.

Proteomics and Protein Expression

overview of cell separation and isolation

overview-of-cell-separation-and-isolation

Methods for Separating Specific Cell Types

Cell separation is used to isolate one cell type from a mixed sample. Early work in this field began in 1964, when S. H. Seal separated large tumor cells from smaller blood cells by filtration. Two years later, Pohl and Hawk showed that different cell types can respond differently to a nonuniform electric field. These findings helped launch modern cell separation methods. Several common techniques are used to separate cells. Filtration removes cells based on size, while density gradient...

cell culture

Growing Cells in the Lab

Growing cells in the lab depends on the right culture type and the right growth conditions. Most vertebrate cells grow attached to a surface as adherent cultures. These cells are grown in flasks and plates that are chemically treated to help the cells attach. Some cells, such as hematopoietic cells, grow in suspension instead. Suspension cultures can be kept in non-treated cultureware with magnetic stirrers or spinner flasks to keep the medium moving. The growth medium is a key part of...

cell lines

How Cell Lines Change and Are Verified

Cell lines are populations of cells grown in vitro that can be subcultured over many generations. Normal cells eventually stop dividing after a set number of cell divisions. This stopping point is called replicative senescence. The division limit is known as the Hayflick limit. Leonard Hayflick described this limit in 1961 after he found that fetal cells in culture could divide only about 40 to 60 times. Telomeres help explain why this happens. Telomeres shorten with each round of cell...

hybridoma technology

Monoclonal Antibody Production with Hybridomas

Hybridoma technology makes monoclonal antibodies in large amounts. Monoclonal antibodies are antibodies that bind to just one antigenic determinant, or epitope. These antibodies are important in research, diagnostics, and disease therapy. The method was developed in 1975 by Georges Köhler and Cesar Milstein, and it received the Nobel Prize in Medicine in 1984 for changing research and treatment. The process starts by fusing cells with different strengths. Common fusion methods include...

tissue homogenization and cell lysis

Lysis Methods for Preparing Tissue Samples

Tissue sample preparation often starts with homogenization and cell lysis. This step breaks tissue structure and opens cells so their parts can be studied. The best method depends on the sample type, the amount of sample, the analyte to be collected, and how sensitive the method is. Tissue homogenization methods are usually grouped into mechanical and non-mechanical methods. Mechanical methods use external physical force to disrupt tissues and cells. These methods may use grinding, shearing,...

subcellular fractionation

Separating Cell Organelles by Centrifugation

Separating cell organelles by centrifugation lets scientists isolate membrane-bound parts of a cell after lysis. The starting material is a homogenate, which is the mixture left after cells are broken open. These isolated fractions help researchers study specific cell parts, measure protein activity in one location, and support some diagnostic tests. Fractionation usually uses centrifugation, which spins the sample to separate its contents. The two main methods are differential centrifugation...

flow cytometry

Flow Cytometry in Cell Sorting

Flow cytometry in cell sorting tracks cells as they move through a laser beam. The instrument measures light scatter and fluorescence, then uses those signals to separate cells based on their properties. It is also used to count cells and study cell populations in detail. The method began in 1934 with Andrew Moldavan, a bacteriologist who counted cells in a flowing capillary system. He pushed cells through a capillary tube and viewed them under a microscope. Later, photometry made it possible...

principles of column chromatography

Column Chromatography: Separation Factors

Column chromatography separates compounds in a sample mixture by passing a solvent through a packed column. The technique was first invented in 1901 by Michael S. Tswett, a Russian botanist, who used organic solvents to separate plant pigments. In 1941, Archer John Porter Martin and R. L. M. Synge improved the method by packing silica gel into a column and separating amino acids with a chloroform-water mobile phase. That work became the first report on column chromatography. Today, column...

types of column chromatography

Column Chromatography for Protein Purification

Column chromatography is used to purify proteins by matching the column material to the sample. The stability and compatibility of the material matter because pH, temperature, and solvent can change how the packed column performs. The right column choice helps improve purification efficiency and can be adjusted for the protein being isolated. Gel filtration chromatography separates proteins by size when the protein’s chemical nature is unknown. It uses matrices such as agarose, polyacrylamide,...

immunoprecipitation

Protein Capture with Antibodies

Immunoprecipitation uses antibodies to capture a specific protein or protein complex from a sample. The protein stays in its native state, which helps scientists study protein-protein interactions, quaternary structure, and larger supramolecular complexes. Several related methods build on this idea. Chromatin immunoprecipitation, or ChIP, is used to study protein-DNA and protein-RNA interactions. Cross-linking immunoprecipitation, or CLIP, is used to find where proteins bind endogenous RNA...

tagging and fusion proteins

Protein Tags for Purification and Detection

Protein tags help scientists isolate and study a specific protein from the other proteins in a cell. The target gene is overexpressed in a suitable host so the cell makes large amounts of the protein of interest. A tag, or label, is added to the gene to create a fusion protein that combines the target protein with the tag. These tags make detection and purification easier. Common tag types include affinity tags, epitope tags, reporter tags, fluorescent tags, and self-splicing intein tags. In...

sds-page

Protein Size Separation by SDS-PAGE

SDS-PAGE separates proteins by size using a polyacrylamide gel and an electric field. It is a type of gel electrophoresis, which moves biological macromolecules through a gel matrix. In PAGE, proteins pass through polyacrylamide gel and can be separated by size, charge, or both, depending on the method used. In native PAGE, intact proteins keep their natural charges and are separated by both net charge and size. In SDS-PAGE, proteins are first denatured and coated with sodium dodecyl sulfate,...

western blotting

Western Blotting for Protein Detection

Western blotting is a protein detection method used in biology and medicine. It helps identify proteins in biological samples and can support disease detection, including bovine spongiform encephalopathy, human immunodeficiency virus, and feline immunodeficiency virus. The process starts with SDS-PAGE, which stands for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This step separates proteins in a sample before they are moved to a membrane. The proteins are then transferred to...

two-dimensional gel electrophoresis

Protein Separation by Charge and Mass

Two-dimensional gel electrophoresis separates proteins by charge first and then by mass. It was first introduced by O’Farrell and Klose in 1975. Because it uses two separating steps, it gives a higher-resolution result than one-dimensional gel electrophoresis. The first dimension uses isoelectric focusing, or IEF, on immobilized pH gradient, or IPG, strips. Before this step, biological samples such as cells and tissues are treated with a buffer that contains urea, dithiothreitol, detergents,...

enzyme-linked immunosorbent assay

ELISA Test Types and Signal Detection

ELISA is an antibody-based test used to detect and measure specific molecules in a sample. It was developed in 1971 by Peter Perlman and Eva Engvall. Unlike western blot, ELISA is done in microtiter plates or in vivo rather than on an absorbent membrane. In ELISA, an antibody has an enzyme attached to its constant region. The variable region stays free to bind a matching antigen. After binding, an enzyme-substrate reaction makes the target visible or measurable. In many tests, the substrate is...

maldi-tof mass spectrometry

MALDI-TOF for Rapid Sample Analysis

MALDI-TOF mass spectrometry analyzes compounds by their mass-to-charge ratio, or m/z. It is a powerful way to detect and separate many chemical and biological samples. A mass spectrometer has three main parts: an ion source, a mass analyzer, and a detector. These parts work together, and the design can change depending on the source and analyzer used. Matrix-assisted laser desorption ionization, or MALDI, is a common ion source in these instruments. It creates soft ionization, which helps...

peptide identification using tandem mass spectrometry

peptide-identification-using-tandem-mass-spectrometry

Reading Peptide Fragments with Tandem MS

Tandem mass spectrometry helps identify peptides by measuring their mass and breaking them into smaller pieces. It is also called MS/MS or MS2. The method uses two mass analyzers to isolate a biomolecule and study its chemical properties. This approach is important in proteomics, the study of proteins on a large scale. Peptides from a complex mixture are often first separated as much as possible after enzymatic digestion. Instruments such as gel electrophoresis or liquid chromatography can...

x-ray diffraction of biological samples

Reading Crystal Patterns with X-Ray Diffraction

X-ray diffraction, or XRD, is used to study ordered structures in crystalline organic and inorganic samples, polycrystalline materials, proteins, carbohydrates, and drugs. The method shows how atoms are arranged in a sample by measuring how X-rays scatter from it. When X-rays hit a sample on a stage, the beams interact with the electron clouds around the atoms. The scattering follows Bragg’s law, and it comes from constructive interference of X-ray waves reflected from internal crystal planes.

applications of nmr in biology

NMR Spectroscopy in Biology and MRI

Nuclear magnetic resonance (NMR) spectroscopy is a useful analytical tool in biology. Researchers have used it for more than 50 years. F. Bloch and E. Purcell developed NMR in 1946 and received the 1952 Nobel Prize in Physics for this work. NMR studies biological macromolecules and small molecules. These include proteins, nucleic acids, lipids, and organic compounds such as pharmaceutical drugs. The technique works by using the magnetic properties of certain nuclei. The basic idea behind NMR...

Proteomics studies the proteins made by a cell type and how those proteins function. A proteome is the full set of proteins produced by a cell type, and proteomics is the large-scale study of that protein set. The term was coined by Mark Wilkins, who described it as the “PROTein complement expressed by a genOME.” Proteomics is closely linked to genomics because genes lead to mRNAs, and mRNAs usually encode proteins. However, not every mRNA is translated into a protein. This means mRNA data can...

key terms and concepts

Explore how proteomics studies protein expression, tissue-specific differences, protein changes after translation, and key tools like mass spectrometry and X-ray crystallography.

Proteomics and Protein Expression | JoVE K12

cell biology

JoVE Core Cell Biology illustrates complex cellular processes in easy-to-understand yet comprehensive animated video lessons. This collection focuses on the essential components and functions of cells, including organelles and membranes, cellular transport, and cell energetics. It is complemented by scientist-in-action videos that demonstrate the application of cell biology in real-world research experiments.

Proteomics and Protein Expression

Description

Transcript

Tags