Biology Reference
In-Depth Information
3
Protein/Peptide Separation and Identifi cation
Two approaches are generally distinguished in the fi eld of pro-
teome analysis a protein based approach (in general referred to as
gel based approach) and a peptide based approach (in general
referred to gel free or as shot gun approach). In the fi rst approach
proteins are separated and quantifi ed. The proteins of interest are
then digested and the resulting peptides identifi ed via mass spec-
trometry. In the second approach, protein digestion precedes the
separation and quantifi cation of peptides. Yates and coworkers
were one of the pioneers to explore the use of two-dimensional
liquid chromatography coupled to electrospray tandem mass spec-
trometry (the Mudpit approach) to realize automated high-
throughput shot gun proteomics [
11
]. A hybrid form where
separation of proteins by gel electrophoresis is followed by the
separation of peptides via liquid chromatography is called geLC.
Almost all proteome studies on orphan species preferentially use a
protein-based approach since all peptide-based strategies have the
disadvantage to lose connectivity between peptides derived from
the same protein [
9
]. The high resolving power of the two-
dimensional protein-based separation technique is of great impor-
tance aiming for an individual protein separation. This assures that
all derived peptides come from the same protein. In the case of
protein separation techniques with a lower resolution (e.g., broad
IPG strips, 1D SDS-PAGE, or geLC-MS) multiple proteins are
digested simultaneously resulting in a more complex peptide pool.
When a combination of masses of possibly non-related peptides is
submitted to a database search, the risk of generating false positive
identifi cations increases. It is therefore crucial that the parent ions
are analyzed further by Tandem mass spectrometry (MS/MS).
MS/MS has been used for decades to obtain structural informa-
tion of (bio)molecules. The main advantage of a protein based
approach is that it can be successfully used for the identifi cation of
protein orthologs based on structural information of the different
peptides. For peptides derived of the same protein, MS/MS gener-
ates sequence specifi c information and the information content of
such spectra in combination with the parent masses (Peptide Mass
Fingerprinting (PMF)) can lead to a successful identifi cation. In
the MS mode, peptides with a high signal-to-noise (S/N) ratio are
selected for further fragmentation. In the case of orphan species, it
is possible that such intense peptides are less or not informative
(i.e., not present in a database and not conserved in a sequenced
species) and classical identifi cation will fail since more homologous
peptides with a lower S/N ratio will not be selected for MS/MS.
However, when the peptide mixture consists of a limited amount
of peptides which are all derived from a single protein, the infor-
mative peptides with a lower S/N ratio have a bigger chance of
being selected for MS/MS, making protein identifi cation more
likely. Unfortunately, almost all software tools are developed to
