3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
Fig. 2. Cbb stained 12.5 % 1-D SDS gel of TRIS extracts and
its AC fractions. Lane 1 – molecular weight markers, Lane 2
– crude TRIS extract (CE); Lanes 3, 4 – Con A non-bound protein
fractions of TRIS extract (NF); Lanes 5, 6 – Con A bound
glycoprotein fraction of TRIS extract (bF)
Table I
Identified proteins from TRIS extract and its AC fractions
s710
on albumin/globulin fraction extracted by TRIS buffer (see
Experimental) that is important for food industry, because the
proteins belonging to this fraction survive malting and brewing
procedures and influence beer quality.
Lectin AC, one of the most effective glycoprotein isolation
methods, was used for other simplification of albumins/
globulins mixture and for the acquisition of fraction containing
a high degree of barley glycoproteins. Since a common
feature of plant seed glycoproteins is the presence oligosaccharide
chains having an affinity for Con A, it was used to
enrich the fraction of barley high-mannose and hybrid-types
N-glycoproteins having affinity for this kind of lectin. The
particular proteins (Con A non-bound fractions) and glycoproteins
(Con A bound fraction) were subjected to SDS-
PAGE. 1-D gel of AC fractions is shown in Fig. 2. Major
bands were cut out (Fig. 2.) and exposed to the trypsin ingel
digestions. Extracted mixture of peptides was subjected
to MS and MS/MS analyses. Subsequent database searching
led to the identification of numerous groups of barley proteins
with molecular masses ranges from about 70 to below
20 kDa. Their summary is shown in Table I.
Conclusions
These experiments confirmed that combination of AC,
SDS-PAGE and MALDI-TOF MS is convenient for the
characterization of barley (glyco)proteins. The developed
procedures can be used for the solution of important industrial
(e.g. brewing) problems.
Indication Indication Molecular Molecular Accession Protein name
of gel spots of gel spots mass (theor.) mass (exp.) number
(extract) (AC fractions) [kDa] [kDa]
– BF66 72.5 ~ 66 gi|421978 BEG1
CE55 nF55 57.9
~ 57
gi|38349539
59.6 gi|10953877
β-Amylase
CE55 BF52 57.7 ~ 57 gi|804656 β-Glycosidase
CE 52 BF52 72.5 ~ 55 gi|421978 BEG1
CE43 nF43 43.3 ~ 43
gi|1310677
P06293
Protein Z serpin
Protein Z
CE36 BF36 72.5 ~ 36 gi|421978 BEG1
– BF32 31.5
~ 31
gi|15824660
25.6 gi|72333
Con A
CE32 nF32 30.0 ~ 32 gi|132577 rRnA n-glycosidase
CE28 nF28 28.5 ~ 28 gi|116316 Endochitinase
CE25 BF25 72.5 ~ 25 gi|421978 BEG1
CE23 nF23 20.0–22.4 ~ 23
gi|439275
gi|4699834
gi|18920
gi|18916
Mixture of
α-amylase/subtilisin
inhibitors
– BF21 72.5 ~ 21 gi|421978 BEG1
CE16 nF16 16.1 ~ 16 gi|439275 CMa-α-amylase inhibitor
CE16 nF16 17.9 ~ 16 gi|452325 CMd-α-amylase inhibitor
– nF16 16.7 ~ 16 gi|6634471 CMe-α-amylase inhibitor
– BF16 72.5 ~ 16 gi|421978 BEG1