3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
P61 ThE PROTEOMIC ANALySIS OF bARLEy
ALbuMINS AND GLObuLINS
MARKéTA LAŠTOVIČKOVá and JAnETTE
BOBáľOVá
Institute of Analytical Chemistry of the ASCR, v.v.i., 602 00
Brno, Veveří 97, Czech Republic,
lastovickova@iach.cz
Introduction
The proteins and glycoproteins play many important
roles not only in nature, but also in technologies (e.g. food
chemistry, medical technologies).
The characterization of glycoproteins is not an easy task
and several techniques have to be used for the resolving of
their total chemical composition. Today, methods including
gel electrophoresis, LC, and MS together with the database
searches have been increasingly used. MS has become a widely
used method in protein analysis after the invention of
MALDI and electrospray ionization techniques 1 .
Proteomics is increasingly used to address questions
of development, physiology and quality of crop plants 2,3 .
Modern analytical techniques are used for the characterization
of cereal seed composition in order to increase basic
knowledge about these plants for economical and nutritional
aspects. One of the most important crops is barley (Hordeum
Vulgare), which is used mainly for malting and animal feed 4 .
The production of (glyco)proteins in barley seed is of particular
interest, because this approach can be utilized to produce
barley lines with improved properties, such as malting quality
due to expression of thermotolerant enzymes 5 .
Experimental
e x t r a c t i o n o f P r o t e i n s
The grains of malting barley (cultivar Jersey) were
obtained from Research Institute of Brewing and Malting
(Brno, Czech Republic). 250 mg of flour was mixed with
2 ml of extraction reagent (TRIS buffer – 0.1 M, pH 7.8 containing
0.5 M naCl; 1 mM MnCl 2 ; 1 mM CaCl 2 ) and shaken
2 h. The suspension was centrifuged (20 min; 14,000 rpm).
The supernatant containing the proteins was filtered using
0.45 µm microfilter (Millipore) and used for the following
experiments.
A f f i n i t y C h r o m a t o g r a p h y ( A C )
A glycoprotein enriched fraction was obtained by Concanavalin
A (Con A) lectin chromatography (Calbiochem).
4 ml of TRIS extract was applied on the column. Purified
glycoproteins were released by 500 mM glucose.
1 D - G E
The proteins were separated by 12.5 % SDS gels. SDS-
PAGE separations were performed on 16 × 14 cm gels (OWL
SEPARATIOn SYSTEMS).The visualization was carried
out by Coomassie Brilliant Blue R-250 (CBB).
s709
I n - G e l T r y p s i n D i g e s t i o n a n d M a s s
S p e c t r o m e t r y
The particular barley proteins and glycoproteins separated
by 1-D GE were subjected to in-gel trypsin digestion 6 .
Peptides, obtained by trypsin digest, were analyzed by MS.
MS experiments were carried out on an Applied Biosystems
4700 Proteomics Analyzer mass spectrometer. α-Cyano-4hydroxycinnamic
acid (10 mg m –1 0.1% trifluoroacetic acid/
acetonitrile (1 : 1, v/v)) was used as a matrix for MALDI-TOF
MS analysis of peptides obtained by trypsin in-gel digestion.
nitrogen was used as the collision gas for MS/MS experiments.
Protein identification was performed by searching the
peptide masses and MS/MS sequence stretches against the
sequence databases using the MASCOT or ProFound search
engines.
Results
This work was focused on the systematic proteomic
study of barley grains. Barley was selected as a model sample
because it contains a complex mixture of proteins and glycoproteins
and has a lot of analogies in other plant materials.
In general, it is not possible to identify the proteins
without previous combination of MS with the separation
techniques. Therefore especial attention was paid to the optimization
of extractions and following separation methods.
The different extraction reagents, selected on the basis of
Osborn’s protein classification, were tested and obtained protein
patterns were compared. Fig. 1. showed noticeable differences
between individual extract. The attention was focused
Fig. 1. Cbb stained 12.5 % 1-D SDS gel of barley proteins
extracted by different extraction reagents. Lane 1 – molecular
weight markers, Lane 2 – aqueous extract; Lane 3 – salt extract;
Lane 4 – TRIS extract, Lane 5 – ethanol extract