Biomedical Engineering Reference
In-Depth Information
a key tool in proteomics research because it can analyze and identify
compounds that are present at extremely low concentrations (as little
as 1 pg) in very complex mixtures by analyzing its unique signature.
There are three parts to a mass spectrometer: the ion source,
the mass analyzer, and the detector. Three broad types of ionization
methods are used in MS, including electrospray ionization, electron
ionization, and matrix-assisted laser desorption/ionization. Analyzers
use dispersion or filtering methods to collect and sort ions according
to their mass-to-charge ratios. Several types of analyzers exist, includ-
ing quadrupole mass analyzers, quadrupole ion trap mass analyzers,
fourier-transform MS analyzers, and time-of-flight mass analyzers, to
name but a few. The ions are then detected by various light or charge
detectors. Frequently, MS procedures are known by the combination of
ionizer and analyzer that is used. For example, matrix-assisted laser
desorption/ionization (MALDI) MS, combined with a time-of-flight (TOF)
analyzer is referred to as MALDI-TOF MS. A critical concern in MS is
that the methods used for ionization can be so harsh that they may gen-
erate very little product to measure at the end. The development of “soft”
desorption ionization methods by John Fenn and Koichi Tanaka, which
allowed the application of MS to biomolecules on a wide scale, earned
them a share of the Nobel Prize in Chemistry in 2002.
MS is used following 2-dimensional gel protein separation for pro-
teomic analysis. It can be used to rapidly and unequivocally identify the
protein in a spot on a 2-dimensional gel, even at low abundance. Mass
mapping is used to identify a protein by cleaving it into short peptides.
The mass spectrometer can then be used to deduce the protein's identity
by matching the observed peptide masses (which are determined with
incredible accuracy) with protein databases. MS has therefore become
a prime method for helping to understand changes in the expression of
proteins in cells during lineage development, in infected cells versus
their normal counterparts, or in transformed cells. While methods to
examine differentially expressed genes have also been developed (see
Chapter 3), the expression of mRNA does not always mean that the con-
comitant protein is expressed, and thus approaches that allow for direct
protein identification are important. In addition to identifying proteins,
tandem MS can be used to directly sequence peptides from proteins
by colliding them with non reactive gas and analyzing the fragmented
ions that are produced. Not only does this provide direct amino acid
sequence information, but also reveals post-translational modifications
such as phosphorylation, sulfination, and the size, complexity can dra-
matically change the function of a protein. Therefore, proteomic analysis
is an indispensable addition to genomic analysis.
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