Biology Reference
In-Depth Information
simple comparison between states A versus B is no longer adequate
to perform functional modeling. Instead, we need high sample
throughput technologies able to identify and quantify as many as
possible proteins of the system. Thus, there is a need for continu-
ous method development and improvement.
Nevertheless, there are obvious strategies to increase sample
throughput as well as number of protein detections. One strategy
is based on the unbiased or untargeted identifi cation and quantifi -
cation of as many proteins possible. In the following this method
is called exploratory protein analysis and is based on recent devel-
opments in proteomics technology called shotgun proteomics.
This is a high sample throughput method in which complex pro-
tein samples from tissue or cells are directly proteolytically cleaved
into small peptides and subsequently analyzed via liquid chroma-
tography coupled to mass spectrometry. These peptides can be
directly quantifi ed without identifi cation using a novel approach
called MAPA (mass accuracy precursor alignment) [
3
]. This allows
the detection of all peptides which are quantitatively changed inde-
pendent whether they are identifi ed by database search. The pros
and cons of such an approach over the classical shotgun proteomics
technique are discussed in the following chapters. Eventually, pep-
tides are identifi ed via their fragment fi ngerprint against genomic/
predicted proteomic databases (see last paragraph “genome anno-
tation”). Protein identifi cation is then based on the reconstruction
from these identifi ed peptides. Compared to classical methods in
proteomics, shotgun proteomics is characterized by a very high
protein identifi cation rate and thus qualifi ed to establish huge
qualitative and quantitative proteome maps for genome-sequenced
but also non-sequenced organisms [
3
,
10
]. Another strategy uses
those proteome catalogues for the design of “proteotypic” pep-
tides of a protein for a targeted analysis. We call this technique
MASS WESTERN because it resembles the Western blot technol-
ogy with the ability to detect proteins in complex matrices after
several preparatory and pre-purifi cation steps for instance using an
SDS-PAGE step [
4
]. The MASS WESTERN has several advan-
tages over a classical antibody-based approach for targeted protein
analysis such as higher specifi city, simultaneous analysis of several
proteins in a complex sample and, of course, no need to produce
antibodies against the target protein. In the next paragraphs we
introduce these techniques associated with multivariate statistics
and a proteomic database.
3
MAPA (
M
ass
A
ccuracy
P
recursor
A
lignment) and ProtMAX
Genomic databases and their corresponding computer-predicted
proteomic databases became essential for proteome science. They
form the bases for protein identifi cation of shotgun proteomics
