Biology Reference
In-Depth Information
search in a non-error tolerant way against a database of known
proteins, which produces low protein scores when several peptide
sequences of the orphans are not identical. EST sequences are
nowadays quite effi ciently and cheaply achieved with the next gen-
eration sequencing techniques and are indispensable.
Depending on the availability of EST sequences, the genome
status of the species under investigation and on the degree of
homology to a model organism, de novo sequencing is essential to
obtain sequence information. Sequence reconstruction of an
unknown peptide based solely on the acquired mass data is referred
to as de novo peptide sequencing. Early de novo sequencing
involved the use of chemical microsequencing using Edman chem-
istry. This method, however, is quite expensive in terms of reagent
cost and suffers from a low throughput and sensitivity. MS based
de novo sequencing is much more sensitive (needs less starting
material). ESI-MS/MS routinely provides more informative MS/
MS spectra than MALDI-TOF MS/MS. Multiple charged peptide
ions tend to fragment more equally across a given sequence than
do singly charged ion species [
12
,
13
]. High precision mass spec-
trometry dramatically improves the performance for de novo
sequencing. The high precision eliminates drastically the number
of candidate peptide sequences that could fi t a tandem mass spec-
trum. Unfortunately, separation of peptides prior to MS/MS is
relatively expensive and time consuming. For these reasons,
MALDI is often preferred in a protein based approach because of
ease of use, speed and the ability to include MALDI spotting in
automated digestion protocols on liquid handling systems. In
addition, a MALDI approach has the advantage that it has the
potential to store temporarily the targets for reanalysis when cer-
tain data are not yet fully explored. We have recently optimized an
automated approach for the derivatization of peptides enabling a
facilitated MALDI based MS/MS de novo identifi cation [
3
].
The protein based 2-DE technique is an excellent choice for pro-
teomics in orphan plants [
9
]. Unfortunately the technique has some
major drawbacks, i.e., it has a very poor performance regarding the
analysis of hydrophobic and basic proteins and is limited in through-
put. A peptide based proteome analysis is the perfect answer to these
limitations. However, a peptide based approach is this feasible for an
orphan plant species? For a review on the analysis of hydrophobic
membrane proteins of orphan species the reader is referred to [
14
].
We will focus here further on a peptide-based workfl ow in general.
As indicated above, proteomics on orphans relies the presence of
EST libraries, on de novo sequencing and on cross-species compari-
son. To increase the chance of identifying conserved peptides, it is
important to ensure a good peptide separation. In this proposed
workfl ow this is achieved by using a RP-RP UPLC system coupled to
a high accuracy mass spectrometer as described by Gilar and colleagues
[
15
]. It is important to build a species specifi c in-house database and
to search this database in a non-error tolerant manner. Subsequently
