My Research on Mass Spectrometry Characterization of Membrane Proteins for Five Levels of Understanding

By Melissa Leyden

UVA ChemSciComm

Primary School:

            Our bodies are made up of many small cells that keep us alive. Each of these cells has even smaller things, called proteins, that do different jobs to keep the cells running. Like a school needing students, teachers, cafeteria people, and janitors to run, these proteins all have different roles in the cell. Proteins are extremely small, so small we cannot see them with our eyes. To see what proteins are made of scientists must use a special tool, called a mass spectrometer. With a mass spectrometer, scientists can see every piece of a protein and what it is made of, which allows them to understand how proteins perform their jobs. Membrane proteins are special proteins used to help cells communicate with each other within our bodies. This makes them a very good target for medicines that could kill cancer. To make the best medicine possible, scientists need to understand how it will work with membrane proteins. Mass spectrometers show scientists what membrane proteins are made of which helps them understand how they will interact with medicine! This will hopefully help doctors and scientists treat cancer in new ways!

High School:

Proteins are more than foods we consume to fuel our bodies. Proteins are also critical molecular compounds made by our cells with important roles. They each have a specific job to help keep cells alive! Proteins can act as bodyguards, store information, and communicate with other cells just to name a few. Membrane proteins in cellular walls are necessary to send and receive signals from one cell to another, making them a great option as a target for medicine for diseases such as cancer. Cancer spreads from cell to cell, so understanding membrane proteins is a great way to make better medicines to treat it, as well as other diseases. To understand how membrane proteins’ function and how medicines impact them, we must know what they are made of. Scientists have very powerful machines called mass spectrometers that can identify the building blocks of proteins called amino acids. Once scientists know which amino acids make a protein, they can better predict how that protein will interact with medicines.

College:

Doctors and researchers are attempting to make new medicines to fight cancer, and some of those medicines target membrane proteins. To make the most effective medicines we must understand how they interact with the target membrane proteins. One way to study this, is to look at the chemical interactions that occur between molecules. Chemistry involves the use of many tools called instruments that help identify different types of molecules. One of these instruments is called a mass spectrometer, an instrument that can be used to identify the structure of molecules. Scientists can use mass spectrometers to identify the amino acid building blocks of membrane proteins. We can use knowledge of the amino acid sequence to predict what chemical reactions may happen between proteins and medicines. New techniques and solutions can be implemented with mass spectrometry for the characterization of membrane proteins, which improve how we can quantify these important interactions. This research will aid in the understanding of many different membrane proteins and the development of effective drugs for many illnesses, including cancer.

Graduate Student in the Discipline:

Protein amino acid sequences can be predicted by DNA sequencing, however, that does not give a full picture of a protein’s final form. After a protein is made, it goes through post-translational modifications that can help a protein perform its job, turn it on or off, or become a form of cellular mutation. Scientists can use analytical chemistry to obtain this biological information. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) allows for the characterization of a protein’s amino acid sequence and any post-translational modifications. This gives a better picture of a protein’s final form, especially those that are a target for drug therapies, such as many types of membrane proteins. Membrane proteins can be digested in a detergent buffer, separated via liquid chromatography, then injected into a mass spectrometer. Each peptide will be isolated by its unique mass-to-charge ratio, then fragmented at the amino acid backbone to allow for the sequence of the peptide to be characterized. With multiple LC-MS/MS analyses and careful data analyses, scientists can put peptides back together like a puzzle, to characterize the entire protein sequence. This characterization aids biological and medical researchers in targeting membrane proteins, giving more accurate information for drug development.

Expert in the Field:

Post-translational modifications can be instrumental for drug design when targeting proteins, as some enable a protein to perform its job or turn it off, leading to cell death. Membrane proteins are no different in this regard and are a popular target for drug therapies for many diseases such as cancer. However, the extremely hydrophobic transmembrane region of these proteins makes them very difficult to digest and analyze in an aqueous solution. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is a very popular analysis for many globular proteins but is not regularly used on membrane proteins because of the requirement of aqueous solutions for protein digestion and analyses. Newly developed detergents can be used with mass spectrometry and allow for LC-MS/MS analyses of membrane proteins. Most specific proteases used in protein digestion and analyses digest at polar residues. This makes membrane proteins bad targets for specific proteases as the transmembrane regions tend to lack these residues. We have developed and optimized an immobilized non-specific enzyme reactor that can digest a protein in a slightly denaturing detergent solution. Utilizing this enzyme reactor and mass spectrometry friendly detergents with LC-MS/MS instruments that allow for multiple fragmentation methods in one analysis we can obtain high sequence coverage of membrane proteins. This has recently been used to characterize a membrane protein and the various post translation modifications that are instrumental to protein function and neuronal survival.