Biomedical Engineering Reference
In-Depth Information
using mass spectroscopy, whose pertinent features I will briefly review, espe-
cially because these features are also important in experimental techniques to
identify protein interactions. For more thorough surveys see (1,24).
The principle of mass spectroscopy is that ions of different mass and charge
travel at different velocity and with different trajectories through an electromag-
netic field. Mass spectroscopy serves to identify the chemical composition of an
unknown sample containing protein or other molecules. A mass spectrometer
consists of three parts: an ionization source, an analyzer, and a detector. The
ionization source is responsible for ionizing the sample, and the analyzer sepa-
rates the ions in an electromagnetic field according to their mass-to-charge (m/z)
ratio. (Ions of the same m/z ratio cannot be distinguished.) The detector collects
the ions and records a spectrum displaying the abundance of ions of a particular
m/z ratio. There are multiple approaches to implementing ionization, analysis,
and detection, as well as to combining them into a spectrometer. I will mention
two approaches that are particular prominent in analyzing proteins.
MALDI-TOF mass spectrometers combine Matrix-Assisted Laser Desorp-
tion Ionization (MALDI) with a time-of-flight (TOF) analyzer. MALDI is a
"soft" ionization method that does not cause fragmentation of molecules in the
sample. In MALDI, a sample is mixed with a matrix compound such as sinap-
inic acid. The mixture of sample and matrix is then exposed to laser light, which
the matrix compound converts into excitation energy that ionizes the sample,
such that it can enter the analyzer. The time of flight analyzer first accelerates
ions in a well-defined electric field and then measures their velocity, which is
characteristic for ions of given m/z. In the typical MALDI-TOF application of
identifying a protein in an organism with a fully sequenced genome, one would
first digest the protein enzymatically into short peptides, and then obtain a mass
spectrum of the resulting mixture of peptide fragments, a so-called peptide map
or mass map. The amino-acid sequence of a short peptide can often be uniquely
determined from its mass. Together with the masses of other peptides from the
same protein, and with the known location of genomic DNA sequences that
have the nucleotide sequence necessary to encode the peptide(s), one can deter-
mine not only the complete amino-acid sequence of the protein, but also the
location of its coding gene.
A second commonly used type of mass spectrometry is tandem mass spec-
trometry (MS-MS). MS-MS uses two analyzers. One method of combining these
two analyzers, daughter ion scanning, is of particular importance for determin-
ing the sequence of peptides in complex protein mixtures. In this approach, ions
pass through a first analyzer, upon which the user (or a computer) selects ions of
a particular molecular weight of interest. These ions are then fragmented by
bombardment with a gas in a process called collision-induced dissociation. (Col-
lision-induced dissociation can be used to determine the amino-acid sequence of
previously selected peptides, where one takes advantage of the fact that peptides
fragment preferentially at their peptide bonds.) The second analyzer then gener-
