In this section, three different mass spectrometry techniques will be discussed. In laboratory research, it is important to understand these techniques.
In MALDI, there is a mixture of two different substances: the matrix and the analyte. The analyte is the substance of interest that needs to be ionized. The matrix is a substance that surrounds the analyte and assists in the ionization process. The matrix and the analyte are mixed together, usually in the liquid phase. The mixture is added, drop wise, to a metal plate. The solvent is allowed to evaporate, and the matrix crystallizes (forms a solid) around the analyte. The metal plate (which is usually stainless steel) is inserted in the instrument. A laser is shot at the sample, and ionization of the analyte is promoted. The ions travel through the instrument, and a mass spectrum is recorded. There are a variety of different types of matrix molecules; usually it is a small, organic acid. One of the most common matrices is called α-cyano-4-hydroxycinnamic acid.
It is widely understood that the matrix promotes the ionization of the analyte. Also, for the most part, only singly charged ions are formed in MALDI. Many times, the most common ion is an analyte molecule with an added or removed proton. We call this the molecular ion. A protonated ion is designated with the symbol [M+H]+. A depronated molecular ion is symbolized with [M-H]-.
The exact mechanism of ionization is still a mystery. It has been theorized that the matrix molecules donate protons to the analyte molecules to form the positive ions.
Electrospray ionization, or ESI, has a very different ionization process than MALDI. The analyte to be studied is dissolved in a solvent, such as an acid in aqueous solution. Ions are formed in the solution, many times protonated by the acid. This mixture is pushed through a small syringe, and it evaporates as it leaves the tip. Essentially, the syringe acts as an aerosol, and the liquid is sprayed into the instrument. Tiny droplets are injected into the instrument, an inert gas, such as nitrogen gas, flows over the droplets. This gas is called a bath gas. The solvent evaporates, and the bath gas keeps the analyte molecules cool. Since these molecules are charged (and all should have the same sign), they repel each other. Eventually, the solvent is completely evaporated, and the analyte molecules burst and fly apart from each other.
At this point, the analyte molecules travel through the rest of the instrument, and are detected. A mass spectrum is produced, which records the percent abundance versus the mass to charge ratio.
Now here is where a difference lies between ESI and MALDI. In MALDI, primarily singly changed ions are formed. In ESI, multiply charged ions are formed. Therefore, a doubly protonated molecule would have z=2 and the symbol for it would be [M+2H]2+.
Ion mobility, or IM, studies another property of charged molecules. There is an ion source in IM, which could be MALDI, ESI, or another technique. Once the ions are formed, they travel through a uniform electric field at atmospheric pressure. The ions are accelerated through the electric field, and are separated as they collide with the atmospheric gas. The time it takes to travel through the electric field is dependent on the cross section of the ions. The larger and bulkier the ions, the longer it will take to travel through the gas. Only a small fraction of the analyte molecules make it through the electric field, and reach the detector. After the electric field has been passed, the ions travel through a "field free" region and strike the detector.
A mass spectrum is produced, as always. However, there is a new piece of information that can be studied. The time taken to travel through the field free region can also be measured and tabulated. The faster ions will have a shorter flight time than the slower ones. If only one type of analyte molecule is studied, there will still be different flight times recorded. The reason for this phenomenon is that a molecule can be oriented in different configurations, and therefore a collection of ions traveling through atmospheric gas will have a variety of different cross sections.
IM allows for scientists to study the configurations and shapes of proteins and peptides.