Wednesday, February 04, 2009

Biochemistry and Mass Spectrometry

 
The following description is from Horton et al.,Principles of Biochemistry 4/e. It explains the use of mass spectrometry in a biochemical context.
Mass spectrometry, as the name implies, is a technique that determines the mass of a molecule. The most basic type of mass spectrometer measures the time that it takes for a charged gas phase molecule to travel from the point of injection to a sensitive detector. This time depends on the charge of a molecule and its mass and the result is reported as the mass/charge ratio. The technique has been used in chemistry for almost one hundred years but its application to proteins was limited because, until recently, it was not possible to disperse charged protein molecules into a gaseous stream of particles.

This problem was solved in the late 1980s with the development of two new types of mass spectromety. In electrospray mass spectrometry the protein solution is pumped through a metal needle at high voltage to create tiny droplets. The liquid rapidly evaporates in a vacuum and the charged proteins are focused on a detector by a magnetic field. The second new technique is called matrix-assisted desorption ionization (MALDI). In this method the protein is mixed with a chemical matrix and the mixture is precipitated on a metal substrate. The matrix is a small organic molecule that absorbs light at a particular wavelength. A laser pulse at the absorption wavelength imparts energy to the protein molecules via the matrix. The proteins are instantly released from the substrate (desorbed) and directed to the detector (see Figure). When time-of-flight (TOF) is measured, the technique is called MALDI–TOF.

The raw data from a mass spectrometry experiment can be quite simple, as shown in the Figure (right). There, a single species with one positive charge is detected so the mass/charge ratio gives the mass directly. In other cases, the spectra can be more complicated, especially in electrospray mass spectrometry. Often there are several different charged species and the correct mass has to be calculated by analyzing a collection of molecules with charges of +1, +2, +3 etc. The spectrum can be daunting when the source is a mixture of different proteins. Fortunately, there are sophisticated computer programs that can analyze the data and calculate the correct masses. The current popularity of mass spectrometry owes as much to the development of this software as it does to the new hardware and new methods of sample preparation.

Mass spectrometry is very sensitive and highly accurate. Often the mass of a protein can be obtained from picomole quantities that are isolated from an SDS–PAGE gel. The correct mass can be determined with an accuracy of less than the mass of a single proton.


©Laurence A. Moran and Pearson/Prentice Hall

No comments :

Post a Comment