Biology Reference
In-Depth Information
Whilst these approaches and the underlying experimental principles were, for a long
time, regarded as the gold standard, they are currently only used in specific cases
and for validation purposes (
Brownridge
et al.
, 2011
), mostly because of restricted
availability of antibodies, the labour intensiveness of the work or, more seriously,
the risk of introducing perturbations that might compromise subsequent systems
biology approaches.
2.2
Mass spectrometry-based absolute quantification
workflows in proteomics
2.2.1
Absolute quantification: Approaches relying on isotope dilution
Overall, mass spectrometry-based absolute quantification strategies are methodolog-
ically based on the concept of stable isotope labelling and the isotope dilution (
Brun
et al.
, 2007
). A defined amount of heavy, isotope-labelled internal standard peptide is
added to a sample of interest. Subsequently, using the known concentration of the
spiked-in peptide as a reference, it is possible to infer the concentration of the probed
sample peptide by comparing the signal intensities of labelled and unlabelled
peptides. Three general approaches have been published for using heavy isotope-
labelled internal standards, the 'Absolute QUAntification' method (AQUA;
Gerber
et al.
, 2003
), the 'protein standard for absolute quantification' method
(PSAQ;
Brun
et al.
, 2007; Dupuis
et al.
, 2008; Jaquinod
et al.
, 2012
) and the
'QconCAT' method (
Beynon
et al.
, 2005; Pratt
et al.
, 2006
).
Historically, the AQUAmethod published by Gygi and co-workers (
Gerber
et al.
,
2003
) was the first to propose the use of synthetic heavy peptides as internal calibra-
tion standards (calibrants). This method of providing internal standards is the most
commonly used for absolute quantification strategies, incorporating synthetic
peptides with high-quality standards/high purities that can be obtained from a variety
of vendors at relatively moderate costs. Despite these advantages, problems may
arise as a result of the tendency of various peptides to stick to surfaces during hand-
ling and by other limitations in the synthesis process of the peptides linked to their
nature of the primary sequences (
Mirzaei
et al.
, 2008
).
An alternative approach to providing a heavy isotope-labelled internal standard is
the use of intact proteins (
Brun
et al.
, 2007; Dupuis
et al.
, 2008; Jaquinod
et al.
,
2012
). This involves expression of the target protein in a heterologous host such
as
Escherichia coli
. The standard protein is labelled by growing the heterologous
host in a heavy isotope-labelled growth medium, and then purifying to homogeneity.
After determination of the heavy-labelled standard protein's concentration by amino
acid analysis, it is used as an internal standard protein by spiking it into the test sam-
ples and using it to determine the absolute abundance of its light (i.e. unlabelled)
endogenous counterpart. This workflow is labour-intensive and heavily dependent
on the proteins to be determined, as the biochemical properties and possible compat-
ibility issues with the host affect the purification and handling of the heterologous
standard protein. Even though this approach provides excellent quantification, the
