Biology Reference
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available from a variety of vendors. In addition to ordering expensive high-quality
peptides, it is also possible to order cheaper, low-quality peptides that are suitable for
optimizing the SRM assays ( Picotti et al. , 2009 ). Aspects to consider when ordering
the peptides are the technical feasibility of chemical peptide synthesis, its length
(optimum length 6-13 residues) and desired functionality (heavy amino acids
incorporated, PTMs) ( Mayya et al. , 2006 ). Additionally, the peptides should be
completely soluble in aqueous solutions and therefore limited in size and the avoid-
ance of excess hydrophobic residues. To ensure that the peptides remain chemically
stable during the analysis under the processing conditions, amino acids that are prone
to oxidation, particularly methionine and cysteine, should also be avoided. Further-
more, consideration should be given to the positioning of the isotopically labelled
amino acid since, to reduce the noise levels of the signals, it is advantageous if both
the precursor and fragment masses of the respective transitions are heavier than their
natural counterparts ( Kirkpatrick et al. , 2005 ). To minimize issues associated with
incomplete resuspension, chemically synthesized peptides should be already
dissolved in an appropriate aqueous buffer system when obtained from the vendor.
This allows the absolute concentrations in solution to be determined by amino acid
analysis.
4.2.1 De novo gene design for QconCAT
Relying on the same basic principles for relative and absolute quantification with a
spiked-in heavy-labelled standard, the difference between absolute quantification
based on AQUA and QconCAT is the source of the peptide standards. Whilst AQUA
peptides are the product of chemical synthesis, the QconCAT peptides are proteo-
lytic products derived from a protein that has been synthesized biologically from
an artificial gene in a bacterial expression host.
In the design phase for such a QconCAT gene, the Qpeptides representing the
proteins under examination (i.e. peptides that are proteotypic) are selected without
the need to consider the restrictions that limit the choice of chemically synthesized
peptides. Selection criteria relate solely to the uniqueness of the protein in terms of
proteotypic peptides, propensity to ionize and detectability in a mass spectrometer.
Nevertheless, limitations that arise as a result of the experimental design, such as the
presence of the amino acids that can be labelled and the avoidance of primary
sequences containing cysteine or methionine that are prone to PTM, still have to
be considered. In order to optimize the synthesis of the QconCAT protein, the ran-
domly concatenated in silico DNA sequences have to be codon-optimized for expres-
sion in the heterologous expression host, usually E. coli . Here, a common limitation
is the formation of unwanted secondary structures in the RNA transcript that might
diminish expression. During the design of the synthetic gene, not only is the sequence
of the Qpeptide gene itself considered, but also additional features such as the inclu-
sion of a start codon, a purification tag (e.g. hexahistidine), protected sacrificial
regions (to avoid exoprotease activity) and quality control peptides. These additional
features allow for affinity enrichment of the expressed protein, and, in particular,
the quality control features add multiple layers of control
in the subsequent
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