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be synthesized onto the labeled standard
peptides. These chemically synthesized internal
standards are usually added after the digestion
step and do not compensate for enzyme diges-
tion ef
a procedure that is impossible using QconCAT
proteins.
For the MRM-MS-based approach of bio-
marker veri
ed as
surrogates for protein targets. Therefore, the
reproducibility and ef
cation, peptides are quanti
ciency of the target proteins in the
sample. The synthetic peptides are generally
straightforward to synthesize and easy to use.
They are normally puri
ciency of the peptides
released from proteins upon digestion are
critical for their quanti
ed to high purity and
cation and could be
a major cause of data variation and discrep-
ancies, as noticed in various studies.
3,42,43
Efforts had to be made to optimize digestion
procedure in plasma for biomarker quantita-
tion.
42,44
It was noted that although an overall
best performing digestion procedure can be
achieved for all proteins in a sample,
quanti
ed by amino acid analysis to assure
quanti
cation accuracy. However, if a large
number of peptides are to be monitored, not
only will the cost of obtaining such high purity
labeled peptide standards become astronomical
but the accuracy of making such a standard solu-
tion mixture from a large number of peptides
could also suffer.
Alternatively, an approach called quanti
the
optimum procedure for best digestion ef
ciency
and reproducibility for any individual protein is
in fact protein dependent.
44
Therefore, a solu-
tion could be using isotope-labeled full-length
recombinant proteins as internal standards for
MRM assays. The recombinant protein stan-
dards assumed to be of similar structure, as
the target endogenous proteins could be added
at the beginning of any sample preparation
and thus compensate for digestion ef
ca-
tion concatamer (QconCAT) was developed in
which an arti
cial protein is expressed using
gene encoding a concatemer of the target
standard peptides in the E. coli system.
39,40
The
QconCAT proteins were reported to be highly
amenable to proteolytic digestion due to their
lack of higher order structure. The result is an
equimolar mixture of the target peptides upon
digestion. QconCAT is a convenient and
economic approach to obtaining an equimolar
ratio of a large number of labeled internal stan-
dard peptides upon digestion. It was evaluated
by Mirzaei et al.
41
to be comparable but not
necessarily superior to the chemical synthesis
method to generate labeled internal standards.
Even though QconCAT does not necessarily
compensate
ciency
as well as variation due to sample prepara-
tion throughout the entire procedure. Isotope-
labeled recombinant proteins have been
explored as internal standards for improved
absolute quantitation accuracy.
45
e
48
Brun
et al.
45
denoted their approach as protein stan-
dard absolute quanti
cation (PSAQ) and
demonstrated that PSAQ outperforms both
AQUA and QconCAT as an internal standard
for quantifying the public health biomarker
staphylococcus super-antigenic toxins in urine
samples (
Figure 5
). Isotope-labeled recombinant
proteins are also the only internal standards
compatible with any sample enrichment, frac-
tionation, and preparation strategies that are
applicable for biomarker veri
for
target
protein
digestion
ef
ciency, due to their completely different
structure, it could still partially compensate for
the digestion procedure, as they are typically
added before the digestion step (as shown in
Figure 4
). However, QconCAT peptides are not
useful
ed peptides as
the protein is expressed in E. coli. In addition,
when multiplexing for biomarker veri
for quantitating modi
cation purposes.
These recombinant proteins are generally
expressed in either cell-free or E. coli systems;
therefore, they are not applicable for quantifying
speci
cation,
a variable amount of internal standards may
need to be added to the different protein mixture
to obtain the best overall quantitation results,
4
c PTMs. As one of the
first applications of
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