Biology Reference
In-Depth Information
relatively high costs and labour-intensive processing precludes its use for large-scale
studies addressing a large number of target proteins.
To address the immense cost and handling difficulties of large-scale AQUA, the
QconCAT technique has been developed as an alternative approach (
Beynon
et al.
,
2005; Pratt
et al.
, 2006
). Here, an artificial gene is constructed allowing for the
expression of a heavy labelled synthetic protein that yields peptides upon tryptic
digestion that match their endogenous counterparts. The advantage of the QconCAT
approach is the concatenation of quantotypic peptides (peptides that are specific for a
protein and may be used for its quantitation) for several target proteins in a single
artificial protein at relatively moderate costs, allowing for multiplex quantification
approaches. Once established and optimized, the production of internal standards is
scalable, providing a robust set of standards for large-scale studies. An additional
advantage of the QconCAT approach is its particular suitability for the study of pro-
tein stoichiometry because labelled quantotypic peptides derived from the QconCAT
protein are present in equimolar amounts.
For both the AQUA and QconCAT approaches, it is crucial to choose appropriate
peptides to serve as internal standards. However, the choice of such peptides can be
difficult, often requiring iterative optimization processes and integration of multiple
levels of information to yield the peptide species that are most appropriate for
absolute quantitation studies (
Mallick
et al.
, 2007
).
2.2.2
Absolute quantification: Approaches relying on label-free
quantitation with best flyer peptides
As alternatives to methods based on the isotope dilution concept, several label-free
quantification methods have been developed for the generation of absolute quanti-
tative data (
Bantscheff
et al.
, 2012
). Instead of spiking-in isotope-labelled standards
into the sample, another characteristic of proteome samples is exploited. It has been
shown that the abundance of a protein in a sample is correlated directly to its signal
intensity in mass spectrometry. This property is used in absolute quantification
methods like label-free quantification with 'best flyer' (Top3;
Silva
et al.
, 2005;
Silva
et al.
, 2006a,b
) and intensity based absolute quantification (IBAQ;
Schwanh¨usser
et al.
, 2011
) approaches.
In the original paper by
Silva
et al.
(2006a,b)
, the strong relationship between
the concentration of a protein and the intensity of the mass spectrometric signal
response for the three most intense tryptic peptides (Hi3) could be used for the
absolute quantification of all of the proteins in a sample (
Silva
et al.
, 2006a,b
). This
workflow provides the option of determining absolute protein abundances in relation
to a spiked-in reference protein that calibrates a universal signal response factor
applied to all proteins. The Hi3 approach can also be used with other data-dependent
mass spectrometric acquisition methods that provide sufficient reproducibility with
respect to peptide sequencing (
Grossmann
et al.
, 2010
). More recently, IBAQ was
introduced as a method that takes into consideration the ratio between the number
of theoretically observable peptides versus the number of actually measured peptides
(
Schwanh¨usser
et al.
, 2011
).
