Biomedical Engineering Reference
In-Depth Information
PI signalling controls fundamental biological processes
including insulin, growth factor and antigen signalling, apoptosis,
cell cycle progression and proliferation. Understanding the roles
of the different PI-modifying enzymes requires methods to quan-
tify their activity status. For PI3K, this is commonly performed by
measuring radioactive de-acetylated PIP3 after metabolic labelling
of cells with radioactive
32
P-ATP. This involves separating the de-
acetylated PI pool by ion exchange HPLC and measurement of
32
P by scintillation or Cherenkov counting in HPLC fractions.
As an alternative to these methods, several studies have
reported the use of LC-MS to quantify PI species. The advantage
of using LC-MS for phospholipid analysis is that by using normal-
phase LC, one does not need to de-acetylate lipids prior to the
erence of enzymes for the phosphorylation of lipids with differ-
ent acyl composition. For example, by using ESI-MS, it has been
demonstrated that there are at least eight different PIP3 species
tion cannot be obtained by other methods for detecting PI species
such as those based on HPLC (briefly outlined above), antibod-
ies or affinity reagents based on lipid-binding domains. Moreover,
LC-MS could in principle be used to profile all PI species in a sin-
gle analysis, thus allowing the quantitative estimation of PI-kinase
and phosphatase activities in a single run.
2.2.Proteomics
One of the first uses of proteomics was as an alternative to cDNA
nale of using proteomics rather than transcriptomics for measur-
ing gene expression is that functional expression of a gene requires
not only its transcription to an mRNA molecule but also the trans-
lation to protein. Initial studies using 2D gels demonstrated that
Later using LC-MS/MS, it was suggested that there was a signif-
icant correlation between mRNA and protein expression in 70%
of instances, thus implying that 30% of expression is controlled at
the level of translation. Therefore, measuring protein expression,
rather than mRNA levels, would be a more direct and relevant
measure of gene expression as this would account for the con-
tribution of both translation and transcription (
see
Chapters 11
,
12
and
13
for protocols for proteome-wide quantification by LC-
MS). Proteomics also has applicability to the analysis of proteins
in blood and other biological fluids whose expression cannot be
analysed by microarrays and to the analysis of proteins in different
cellular compartments (see below).
It is often argued that gene expression microarrays offer
a more comprehensive picture of gene expression than do
2.2.1.Protein
Expression
Search WWH ::
Custom Search