5.6: Proteomics

Biotech Focus

In 1986, Alex Ullrich, a scientist working for the biomedical company Genentech, identified the protein known as the Human Epidermal Growth Factor Receptor 2 (HER2) protein. Using mass spectrometry and protein profiling, researchers measured the overexpression of HER2 in certain breast cancer patients. In the mid-1990s, Dennis Slamon and his colleagues at UCLA, using proteomic techniques, developed trastuzumab (Herceptin), a monoclonal antibody that specifically inhibits HER2 activity. The clinical introduction of this drug has drastically improved survival rates for HER2-positive breast cancer patients. Proteomics-based assays are now used in clinical diagnostics to determine HER2 expression levels in breast cancer tissues, helping guide personalized treatment strategies.

Introduction

Proteomics is the large-scale study of proteins. Proteins are produced based on genetic information encoded in genes, but unlike genes, protein expression, structure, and function can vary greatly under different conditions. The goal of proteomics is to understand the proteome, the full set of proteins expressed in a biological sample, such as a cell or tissue. Unlike the genome, which is relatively stable, the proteome is highly dynamic, with protein expression levels, modifications, and interactions constantly changing. Therefore the proteome is the full set of proteins expressed at a specific time. Proteomics complements genomics by showing how genetic information is translated into functional proteins and how these proteins interact with one another to form complex networks within a cell. By studying these networks, researchers can identify protein complexes and elucidate their roles in signaling pathways and cellular functions. Through proteomic research, scientists can identify protein biomarkers and pathways that drive disease progression, identify potential drug targets, and optimize treatments and reduce side effects. Outside of medicine, proteomics can determine how organisms respond to environmental changes or stressors, by studying changes in protein expression and modification.

Concept in Action:

Video: Proteomics

Learning Objectives

Proteomics studies the structure and function of proteins over a large-scale. At the end of this section, you will be able to:

• Explain mass spectroscopy
• Explain 2D gel electrophoresis
• Explain liquid chromatography
• Explain protein microarrays

The Techniques of Proteomics

The techniques of proteomics can identify the proteins present in a sample, compare protein profiles between different samples, and study protein-protein interactions. Key approaches in proteomics include:

1. Mass Spectrometry (MS)
2. 2D Gel Electrophoresis
3. Liquid chromatography
4. Protein Microarrays

Mass Spectroscopy

Mass spectroscopy (MS or "mass spec") is a cornerstone method for identifying and quantifying proteins by analyzing their mass and structure. Using mass spec, researchers can also learn about post-translational modifications, such as phosphorylation, methylation, or glycosylation, that occur after proteins are synthesized. These modifications are key to protein function and regulation, and proteomics allows researchers to study these modifications and understand their impact on cellular processes.

In mass spec, proteins are digested into peptides that are separated by chromatography and then ionized to convert them into charged particles (Figure \(\PageIndex{1}\)). These ionized peptides are separated based on their mass-to-charge ratio (m/z ratio) using an instrument called a mass analyzer. A detector within the mass analyzer measures the m/z ratio and abundance of each ion, creating a mass spectrum. The resulting spectrum for a peptide can then be analyzed to determine it's identity, which in turn identifies the protein from which it originated.

Figure \(\PageIndex{1}\): The steps of mass spec analysis of the proteome. (1) Digestion of proteins: Proteins are isolated from a biological sample (cells or tissues). The protein mixture is digested into peptides. (2) Separation and ionization of peptides: The peptide mixture is subject to liquid chromatography to separate the peptides which are then ionized by methods such as electrospray ionization to form charged ion-peptides. (3) Ion-peptide analysis by mass analyzer: The ion-peptides are analyzed in several steps by a mass analyzer that determines the mass-to-charge (m/z) ratio and abundance for each peptide. (4) Production of the mass spectrum: The data is compiled into a mass spectrum graph where the identity of each peptide can be determined. (The Steps of Mass Spec by Patricia Zuk, CC BY 4.0; adapted from Mass spectrometry protocol by Phillip Hupe CC BY-SA 3.0 and Mass spec brassicasterol by Smmudge, CC BY-SA 3.0)

Two-Dimensional Gel Electrophoresis

Like mass spec, two-dimensional gel electrophoresis (2D-GE) separates proteins based on their size and charge. 2D-GE is often used in the study of post-translational modifications of proteins owing to its high level of resolution. In the first step, proteins undergo isoelectric focusing, which is separation based on their on their isoelectric point (pI), the pH at which the proteins net charge is 0. Following this, the proteins are then separated by molecular weight. The separated proteins are visualized using conventional stains like Coomassie Blue or and can be extracted from the gel for further analysis by mass spec.

In 2D-GE, proteins are denatured and then applied to a gel strip containing a pH gradient. An electric field is applied, and proteins migrate to their isoelectric point where they "focus" into tight bands. The gel strips can be loaded onto polyacrylamide gels for conventional SDS-PAGE analysis (Figure \(\PageIndex{2}\)). While 2D-GE can separate thousands of proteins based on their pI, the process is laborious and time consuming, making mass spec a more desirable option for labs that can afford the equipment.

Figure \(\PageIndex{2}\): 2D Electrophoresis. 2D gel electrophoresis separates proteins by size and charge using two "dimensions". First Dimension - Separation by Isoelectric point (blue box at left in figure): A protein sample (black oval) is applied to a gel strip that has a pH gradient (pH 10 at top of strip, pH of 2 at bottom of strip). An electric field results in isoelectric focusing of proteins within the gel strip. During focusing, each protein in the sample will migrate from the cathode at the top of the strip towards the anode at the bottom of the strip and stop at their isoelectric point (IP), forming a tight band. At the end of isoelectric focusing, the gel strip will contain multiple proteins, each at their individual IP (colored bands). Second Dimension - Separation by Molecular Weight (green box at right in figure): The gel strip from the first dimension is laid on top of an polyacrylamide gel and an electric field applied. The proteins will exit the gel strip and enter into the polyacrylamide gel. Once in the gel, the proteins will migrate based from the cathode at the top of the gel towards the anode at the bottom of the gel and stop at a position in the gel based on their molecular weight (colored dots). (2D Electrophoresis by Patricia Zuk, CC BY 4.0; Created in BioRender. Zuk, P. (2025))

Chromatography is a widely used technique for separating, identifying, and purifying the components of a mixture, such as proteins (Figure \(\PageIndex{3}\)). There are numerous types of chromatography, including liquid, gas, and thin-layer chromatography. Proteins are often separated using liquid chromatography. To separate proteins using liquid chromatography, the sample is dissolved in a liquid phase called the mobile phase. The mobile phase is then applied to a stationary phase - a solid (or liquid-coated) material that has been fixed into a column - and then allowed to move through the stationary phase. Because of their unique physical and chemical properties, each protein in the sample will interact differently with the stationary and mobile phases. This will result in different rates of movement for each protein through the stationary phase, separating the sample into individual proteins. Depending on the type of stationary phase used, proteins can be separated based on their charge, size, hydrophobicity, isoelectric point, or through their interaction with a ligand attached to the stationary phase.

Figure \(\PageIndex{3}\): Liquid chromatography can separate proteins. Separation of a protein mixture by column chromatography is based on the movement of a liquid, mobile phase through a stationary, solid phase that is formed into a column. The protein mixture is dissolved in the mobile phase (also called the sample eluent) and applied to the column. A stopcock at the bottom of the column is opened to allow for mobile phase migration down the column. As the mobile phase moves through the stationary phase (shown with an arrow), it carries the protein sample with it, resulting in the separation of individual proteins within the column (separated components). (Protein chromatography by Patricia Zuk, CC BY 4.0)

Protein Microarrays

The protein microarray is used for high-throughput screening of proteomes. Like DNA microarrays, the protein microarray is made of a solid support, usually a glass slide, known as a chip. An array of "capture proteins" (e.g. antibodies, antigens, peptides, full-length proteins) are bound to the chip. Sample proteins are labeled with fluorescent probes and then applied to the chip. The capture proteins "capture" the labeled sample proteins by binding them. Those proteins bound to the chip can then be detected and quantified using their fluorescent signal. Using the appropriate protein microarray, the proteins found within a proteome can be tested for their function, expression level, or presence/absence of modifications through their interactions with the proteins on the microarray. Protein microarrays have been key in the study of antibody-antigen interactions, inflammatory mediator production, detection of infectious diseases, and identification of disease biomarkers.

Key Concepts

Proteomics provides a deeper understanding of cellular function, bridging the gap between genetic information and biological processes, making it essential for fields like molecular biology, medicine, and biotechnology.

Some important concepts to remember are:

• proteomics studies the structure, function, and modifications of proteins using techniques such as:
  • mass spectrometry
  • 2D gel electrophoresis
  • chromatography
• the study of the proteome is critical in the construction of biochemical pathways that underlie normal cellular processes and disease like cancer, cardiovascular disease, or neurodegenerative conditions
• proteomics has led to the identification of numerous disease biomarkers and the development of therapeutic drugs
• proteomics can measure drug effects on the proteome to optimize treatments and reduce side effects
• proteomics can reveal how organisms respond to environmental changes, such as temperature shifts, pollutants, or pathogens, by studying changes in protein expression and modification

Glossary

Biomarker – a protein or molecular signature that indicates a biological state, such as disease or infection

Chromatography - a lab technique for purifying compounds in a mixture based on differences in their physical and chemical properties; main types include liquid, gas, thin-layer, and paper chromatography

Isoelectric focusing - the separation of proteins using their isoelectric point

Isoelectric point (pI) - the pH at which a proteins charge is "0"

Liquid chromatography - a separation technique based on the movement of a sample within a mobile phase through a stationary phase

Mass spectroscopy (i.e. mass spec) - an analytical tool useful for measuring the mass-to-charge ratio (m/z) of one or more molecules, like a protein, present in a sample

Mass spectrum - a plot of the relative abundance of a molecule, like a protein, as a function of its m/z ratio

m/z ratio - the mass-to-charge ratio

Protein microarray - a solid surface onto which thousands of different proteins are immobilized; allow researchers to study multiple proteins simultaneously

Proteome - the complete set of proteins expressed by a cell

Proteomics - the study of the proteome

Two-dimensional (2D) gel electrophoresis - a technique that separates proteins first based on their isoelectric point, then their molecular weight