3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
P62 COMPARATIVE STuDy OF ThE PROTEIN
COMPOSITION INDuCED by MALTING
bARLEy OF TwO VARIETIES
MARKéTA LAŠTOVIČKOVá, MARTInA ŠOPíKOVá
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 selection of barley cultivar has great importance for
the malting and brewing industries, as well as for breeders,
because malting properties and resistance of barley plant to
fungal or viral diseases is cultivar-dependent. Since protein
content is one of the most important barley character related
to malt qualities, proteomics is ideal tool for both differentiation
of barley cultivars and description of malting course.
Proteomic analysis usually includes some separation technique
connecting with MS and the database searches. SDS-
PAGE has been commonly used as a separation step before
MS analysis of proteins. Moreover, it seems to be convenient
tool for the study of qualitative differences between barley
cultivar. 1–3
The aim of this study was the characterization of protein
changes and the identification of major proteins that undergo
some modifications during malting process. For these experiments
two barley cultivars were selected.
Experimental
E x t r a c t i o n o f P r o t e i n s f r o m B a r l e y
G r a i n s
Barley grains in different malting degrees (cultivar “Jersey”
and KM1910) were obtained from Research Institute of
Brewing and Malting (Brno, Czech Republic). 50 mg of sample
was mixed with 1 ml of deionized water and shaken 1 hr.
The suspension was centrifuged (10 min; 13,000 rpm). The
supernatant containing the proteins was lyophilized.
P r o t e i n S e p a r a t i o n
Lyophilized extracts were resuspended in 150 μl sampling
buffer (Laemmli Sample Buffer (Bio-Rad) with βmercaptoethanol,
19 : 1) and denaturated (10 min, 95 °C).
SDS-PAGE separations were performed on 15 % TRIS-HCl
(Bio-Rad) or gradient (4–20 % TRIS-HCl, Bio-Rad) gels.
The visualization was carried out by Coomassie Brilliant
Blue (CBB) R-250, CBB G-250 and silver staining.
P r o t e i n I d e n t i f i c a t i o n
The selected proteins separated by 1-D GE were digested
in-gel by chymotrypsin 4 . Obtained peptides were analyzed
by MALDI-TOF MS (Applied Biosystems 4700 Proteomics
Analyzer). α-Cyano-4-hydroxycinnamic acid (CHCA;
10 mg ml –1 0.1% trifluoroacetic acid/acetonitrile (1 : 1, v/v))
was used as a MALDI matrix. Air was used as the collision
gas for all MS/MS experiments. Protein identification was
s712
performed by searching the peptide masses and MS/MS
sequence stretches against the sequence databases using the
MASCOT search engine (database nCBInr).
Results
P r o t e i n C h a n g e s D u r i n g M a l t i n g
Water-soluble proteins were extracted from barley
mature grains, grains after 1–5 days of malting and from green
and ready malt. 1-D GE was performed to obtain a complex
characterization of changes of protein profiles during malting.
The gradient gel (4–20 %) was used to acquire the results
covering the largest interval of molecular masses. Since the
demand for a protein staining (sensitivity, compatibility with
MS) is one of the most important tasks in GE, especial attention
was paid to the selection of protein visualization. Two
types of CBB (R-250 and G-250) and silver staining were
compared (data not shown). The best results were obtained
with CBB-G of which using the differences between protein
patterns during malting were distinct (Fig. 1.).
A visual inspection of gradient gel confirmed significant
modifications observed after three days of malting at minimal
five protein bands at molecular masses about 53 kDa, 43 kDa,
26 kDa, and 23 kDa (see arrows in Fig. 1.). The major changes
were noticed at the band with molecular mass about
43 kDa. Therefore it was cut out, digested by chymotrypsin
and obtained mixture of peptides was subjected to MS and
MS/MS analyses (Fig. 2. a, b). Database searching revealed
its identity as Protein Z (gi/131091). This protein is important
for the brewers, because due to its posttranslational modifications
(glycations) influence the sensory and technological
properties of beer (e.g. foam).
P r o t e i n P a t t e r n f o r D i f f e r e n t
B a r l e y C u l t i v a r s
Extracts from mature grains and malt of two different
varieties, Jersey and KM, were separated via 15 % 1-D SDS-
Fig. 1. Cbb-G stained gradient 1-D SDS gel of protein extracts
from mature grains (Lane 2), one day malting grains (Lane 3),
two days malting grains (Lane 4), three days malting grains
(Lane 5), four days malting grains (Lane 6), five days malting
grains (Lane 7), green malt (Lane 8) and ready malt (Line 9).
Molecular weight markers are in Lanes 1 and 10