Biology Reference
In-Depth Information
The idea of libraries of millions of peptides for the capture of
protein from a crude extract with the aim of reducing the dynamic
concentration range is relatively recent [ 31 ], but a large number of
applications have been described with a variety of protein extracts
and biological fl uids. Several recent reviews [ 32 , 33 ] illustrate them
and demonstrate the excellent results that can be obtained with
this technique and the unique increment in the detection ability.
According to the protein desorption method (single or
sequenced) or according to the type of eluents used, certain types of
analytical methods can be directly applied. For instance when the
desorption is produced by SDS Laemmli buffer at boiling for 5 min,
SDS-PAGE is directly applied [ 34 ]. Alternatively when thiourea-
urea-CHAPS (TUC) solution is used 2D electrophoresis can be
directly used with no preliminary treatment [ 35 ]. In another instance
the captured proteins, still on the beads, can be directly digested by
trypsin and the peptides directly used for multidimensional protein
fractionation and identifi cation via mass spectrometry [ 36 ].
Plant protein extracts are not always dominated by a single or
a few largely dominant proteins like human serum or red blood cell
extracts; however, there are cases where one protein largely domi-
nates the protein content. This is the case of RuBisCO (ribulose-
1,5-biphosphate carboxylase/oxygenase) that accounts for more
than 40 % of the total protein amount in leafs [ 37 ].
The poor protein content of plant extracts frequently associated
to undetectable very-low-abundance proteins and the dominance
of few very concentrated proteins justify adapted protocols for pro-
tein sample extracts to facilitate the analysis of plant proteomes.
2
Materials
Phosphate-buffered saline, ammonium persulfate, protease inhibitor
cocktails, sodium chloride, urea, thiourea, CHAPS, acetic acid, citric
acid: All these chemicals are from regular suppliers such as Sigma-
Aldrich, MO, USA. Plastic labware is from Pierce Biotechnology,
CO, USA. Combinatorial hexapeptide ligand library (ProteoMiner)
is from Bio-Rad Laboratories, CA, USA ( see Note 1 ). Vortex and
benchtop centrifuge are from Thermo Fisher Scientifi c.
3
Methods
3.1 Tissue Extraction
and Pretreatments
Since soluble protein concentration is generally very low in differ-
entiated plant tissues [ 4 ] and a number of undesired material is
present, before entering the process of proteome studies several
points have to be considered. Plant cells are rich in proteases, which
require the presence of inactivating agents, and rich in polysaccha-
rides (lots of them polyanionic) interacting directly with CPLL and
 
Search WWH ::




Custom Search