Biology Reference
In-Depth Information
quantification steps to facilitate the precise determination of protein concentration,
for example, by comparison against a universal peptide standard or quantification by
immuno-affinity methods that target specific peptides found on the QconCAT pro-
tein. An excellent protocol that includes the considerations necessary for the design
of a QconCAT gene was published by
Pratt
et al.
, (2006)
.
4.2.2
Expression, purification and usage of the QconCAT protein
Following the design phase of the synthetic QconCAT gene, the cognate protein is
expressed and, following affinity purification and the determination of its concentra-
tion, is ready to use. The latter two steps are crucial since a high degree of purity and
the precise determination of concentration are necessary for the optimal quantifica-
tion of the proteins of interest. Furthermore, as the Qpeptides are generated in equi-
molar proportions by proteolytic cleavage (at least in theory), a thorough revision of
the proteolytic digestion efficiency of the QconCAT protein and the proteome sam-
ple must be determined. This will ensure a bias-free analysis without an underesti-
mation of the proteins of interest. This is particularly important since when the
Qpeptides are randomly concatenated in the synthetic protein, the natural context
of the primary amino acid sequence is abolished. As a result, it is commonly assumed
that QconCAT proteins do not form specific secondary structures leaving the proteo-
lytic cleavage sites completely accessible. Consequently, the complete proteolytic
cleavage of QconCAT proteins is generally more efficient than that of their natural
counterparts. This issue needs to be addressed by adopting specific digestion proto-
cols that completely denature the native and QconCAT proteins and this issue should
ideally be addressed in every new QconCAT-based study (
Pratt
et al.
, 2006; Rivers
et al.
, 2007; Mirzaei
et al.
, 2008
).
4.3
Quantitative SRM assays based on heavy-labelled peptides
With a source of heavy-labelled peptides finally to hand, it is relatively straightfor-
ward to check the peptides for their fragmentation patterns, retention time and
instrument-specific SRM assay optimization. The parameters that need to be opti-
mized include the choice of best fragments, determination of the prevalent charge
state of the precursor, optimization of collision energies and other, vendor-/instru-
ment-specific parameters. Rather than describing the SRM optimization in detail,
readers are referred to excellent existing protocols that provide technical guidelines
for setting up new SRM assays (
Lange
et al.
, 2008; Gallien
et al.
, 2011; Kettenbach
et al.
, 2011
).
Regardless of the origin of the heavy-labelled internal standard peptides for
SRM, optimization has to be completed prior to starting the actual analysis. In quan-
titative SRM assays, the optimized and scheduled transitions for the peptides under
examination are combined with the methods associated with the mass spectrometric
instrument to ensure that all transitions are determined with the maximum number of
data points (at least 8-10), minimalizing the number of repeated LC-MS runs. In
order to derive quantitative data from the signal intensities of the endogenous peptide
