Biology Reference
In-Depth Information
quantifi cation. The incorporation of a HP internal standard is the
only approach that enables absolute quantifi cation. The peak area
of the HP can be used to normalize data across multiple samples
(by dividing each individual peak area of a sample by the peak area
of the HP of that particular sample). Since a known amount of HP
is spiked into the sample, the peak area of the HP can be compared
to the mean and standard deviation of its calibration curve at the
given concentration. Sample handling and or technical aberrations
can thereby be detected. Subsequently, the measurement can be
repeated or a correction factor applied. The discrete steps to set up
a Mass Western experiment are described as follows.
3.2 Choice of
Proteotypic Peptides
When setting up a Mass Western using the experimental approach,
all identifi ed peptides of the target protein are considered as puta-
tive candidates. The amino acid sequences of each peptide can be
BLASTed against the proteome of the organism, in order to distin-
guish proteotypic peptides (other tools such as Skylineare available
as well). Sequences containing missed cleavage sites should be
avoided, due to the potential occurrence of proteolytic peptides
consisting of partial sequences of the targeted peptide. Sub-
sequently, the total amount of the targeted peptide will decrease
and thus the accuracy and sensitivity of the experiment. Methionine
should be avoided, since it is prone to oxidation. Cysteine residues
tend to build disulfi de bridges and can result in dimers or cycliza-
tion, though alkylation can counteract this problem. Generally,
reproducibly identifi able proteotypic peptides are very promising
candidates for a Mass Western experiment. Peptides with the high-
est ionization effi ciency (and thus the most intense signal), and
best chromatographic peak shape should be chosen.
When approaching a Mass Western experiment theoretically,
the putative list of proteolytic peptides, resulting from in silico
digestion of the protein of interest, needs to be reduced to proteo-
typic candidates. This can be performed by BLAST, as previously
mentioned. Further selection criteria are as follows: Peptide length
should preferably be between eight and twenty amino acids.
Proteolytic effi ciency should be as high as possible and can be esti-
mated by tools such as Peptide Cutter (
http://web.expasy.org/
peptide_cutter/
). N-terminal Glutamine has a tendency to form
cyclic peptides. Low coupling effi ciency due to the hydrophobic
and steric characteristics of Tryptophan can pose problems when
synthesizing peptides. Generally hydrophobicity and thus solubility
of the peptides needs to be considered not only concerning synthe-
sis, but also concerning extraction, digestion and resuspension of
proteins/peptides. Sequences containing Proline can produce
multiple chromatographic peaks due to enantiomers, but are also
known to easily and prominently fragment in MS/MS experi-
ments. Furthermore, potential Post Translational Modifi cations
(PTMs) should be considered. To the best of the author's
