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necessary in cases where closely related proteins do not result in
proteotypic peptides using a single protease.
Many of the disadvantages of non-targeted expeditions tracking
down individual peptides in fractionated cell lysates or diffi culties
in synthetic peptide selection can be overcome by in vitro tran-
scription/translation of the proteins of interest. On the genome
scale this approach might be ambitious; however, individual labo-
ratories typically are interested in networks containing hundreds of
proteins, making in vitro transcription/translation of proteins for
generating SRM assays a viable option [ 17 ].
In vitro transcription/translation will generate whole proteins,
which can be digested by various proteases; hence, a switch in pro-
tease requires only another protein digest. The protease effi ciency
is considered in the context of the protein, e.g., noncanonical pep-
tides generated are considered for SRM assay development.
Peptides of the digested protein are all analyzed in an LC-MS/MS
experiment and best performing peptides can be selected for sub-
sequent SRM assay conformation. Best performing peptides are
often referred to high fl iers and frequently results in 10 times more
signal than other peptides of the same protein present in equimolar
amounts [ 17 ]. The proper choice of high fl ier peptides can make
the difference between detecting the peptide in a complex mixture
of whole cell lysates, or not.
In vitro transcription/translation systems were described for
Escherichia coli [ 18 ] and are commercially available. Further, cell
free transcription/translation systems are available using wheat
germ extract [ 19 , 20 ]. Already the comparison between prokary-
otic and eukaryotic in vitro translated proteins might give insights
into possible post-translational modifi cations (PTMs) of proteins
[ 21 ]. Unexpected PTMs are one of the causes in silico search algo-
rithms do not attribute a spectrum to a certain peptide. Also, as
outlined below, a less complex sample is very valuable tool to deter-
mine signal interferences concerning certain SRM transitions.
In closing, in vitro transcription/translation of proteins might
diverge from conventional large scale SRM assay development
[ 16 ], however, in a more targeted analysis of the dynamic pro-
teome [ 22 ] together with the fl exibility of protease treatment [ 23 ]
favors the whole protein synthesis.
2.2.4 Synthesized
Proteins
3
Notes
1. General Considerations for Peptide Selection
Besides favorably LC and MS properties of the analyte, the
unique nature of the peptide should also be considered.
Unique sequences are peptide sequences that are found only
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