Biology Reference
In-Depth Information
4
Note
Although this chapter is mainly devoted to 2-DE, a similar consid-
eration can be done for label-free approaches, as follows:
Label free LC-MS (ion count
).
Consider only the significant peptide and protein hits for
quantitation. A general rule for defining a significant hit
cannot be given, since it depend both of the quality of the
sample, the employed database, and the precision of the
mass spectrometer. For example in a Orbitrap instrument,
analyzing samples from a fully sequenced organism, using the
specific protein database and the 6-frame translation of the
genome, and employing SEQUEST algorithm a significant hit
can be defined by: (a) at least two peptides with a XCorr > 1.75
or 2.0 (for ions with charge +2 or +3, respectively); (b) only
one peptide with XCorr +0.3 greater than charge state are
sufficient [
18
,
19
]. The determination of protein scores for
orphan species is tricky and dependent of the number and
length of the sequences available in the database [
20
].
●
Label free based quantitation of LC-MS samples a NSAF
[
21
].
●
( /)
( /)
PSM
PSM
L
(
NSAF
)
=
k
k
∑
1
in which the total number spectra counts for the matching
peptides from protein
k
(PSM) was divided by the protein's
length (
L
), then divided by the sum of SpC.
N
L
i
i
Acknowledgements
L.V.'s work was generously supported by a Marie Curie IEF Grant
(FP7-PEOPLE-IEF, European Union).
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