Weighing the Molecules of Life

Margareta Ramström
Department of Analytical Chemistry
Uppsala University
March 2002

Large molecules, proteins, are involved in all the important processes in the body. Hence, it is of great interest and importance to study these molecules, to learn about their functions, and their locations within us. In my research project, in analytical chemistry, I try to detect proteins in different body fluids by weighing them using a mass spectrometer.

In all parts of the human body, as well as in all other organisms, there are large molecules, called proteins. The proteins have important functions that are essential for living, e.g. to transport other molecules to different parts of the body or to enhance biochemical reactions. If a protein is absent, or if the level is reduced or increased, this can cause serious diseases. Studies of these molecules are thus indispensable for answering many questions concerning the human body. Proteins are built up from smaller units called amino acids. There are 20 different amino acids that can be combined to form macromolecules, in the same way as all English words can be written using the 26 letters. The sizes of proteins vary, but most of them contain over 100 amino acids. The order of amino acids determines the chemical properties and the folding of the proteins.

In our laboratory, chemists and physicists collaborate in studying proteins by mass spectrometry. A mass spectrometer is an instrument that can be compared to a scale, but instead of weighing apples and pears, this scale gives the weight, the mass, of molecules. There are different sorts of mass spectrometers, but in the one we use, a so-called FTICR, the molecules are transferred into a strong magnetic field. Charged molecules will start to rotate in this field, and depending on their mass and charge, they will rotate with different frequencies. It is possible to tell the mass very accurately using this technique. From the mass spectrometer we get a mass spectrum, containing all detected masses.

If we know the amino acid composition of a protein, we can also calculate its mass, and compare the known mass to the mass we get from the mass spectrometer. If they agree, we have found a candidate protein in the sample. However, it is possible that some proteins are modified. They may, for example, have taken up an oxygen atom. The modifications change the mass of the molecule. Some proteins are also too large to be detected in the mass spectrometer. One common way to solve these problems is to cut the proteins using another protein, which can be seen as a pair of scissors that cuts the protein only at specific positions, so that we still know what masses to expect afterwards. Then we end up with smaller parts of the proteins, peptides, to identify. These peptides will have reasonable sizes, and a modification will only change the mass of the peptide where it is located. If we find several different peptides from the same molecule, the protein is identified.

In my research, I try to develop a better method for studying proteins in biological samples such as plasma from blood and cerebrospinal fluid. These samples contain an enormous amount of proteins and other components. There is too much information for the mass spectrometer to analyze, if all molecules are present at the same time. In my approach I try first to separate the peptides, whereby I send them directly into the mass spectrometer. I record a mass spectrum every 10th second during the separation, which means that I tell the mass spectrometer to give me the masses of all peptides that entered during 10 seconds. In this way, I get many mass spectra from the same sample, but each spectrum gives me clear information. I am collaborating with a group at the University hospital, and together we compare the protein repertoire in cerebrospinal fluid from persons suffering from a neurological disease, and from healthy persons.

Researchers that are studying proteins to try to better understand certain diseases or to develop new drugs are in need of better tools for their analysis. I hope that the method I develop can be useful for them, and also complementary to those already existing.