Practical Report on Proteomic Analysis of Grapes - University

This practical report details a proteomic analysis of grapes, focusing on the changes in protein profiles during the ripening stages. The study investigates the correlation between physiological and biological changes with proteomic profiles, particularly examining the role of proteins like chitinases and thaumatin-like proteins (TLPs) in both the skin and pulp of grape berries. The report outlines the materials and methods used, including protein extraction from grape berry skin and the application of 2-DE-based DIGE for proteome analysis. The research aims to understand how grape networks respond to external stress and the factors influencing berry development, with the ultimate goal of identifying inter-seasonal differences and understanding the molecular processes involved in organic and sugar metabolism. The report also highlights the importance of understanding protein haze formation in wine and the application of proteomics in analyzing the stress response and development of grape berries.

Practical Report on Proteomic Analysis of Grapes 1
PRACTICAL REPORT ON PROTEOMIC ANALYSIS OF GRAPES
By Name
Course
Instructor
Institution
Location
Date

Practical Report on Proteomic Analysis of Grapes 2
Practical Report on Proteomic Analysis of Grapes
Abstract
Grapes are one of important crops in the world economically wise. The quality of grapes
is determined by components such as flavors, sugars and tannins which are usually accumulated
during the different development stages of the berry. In order to understand the changes that
occur during berry development and the different ripening stages we have to correlate the
physiological and biological changes to the proteomic profiles. During the green development
carbohydrates profiles showed consistence with net sucrose to malate conversion. The
abundance of soluble acid invertase and sucrose synthase was correlated to the beginning of
ripening. Protein hazes may form in finished white wine and this is a crucial fear to wine making
companies (Tian, 2015). The protein hazes mainly consists of chitinases and thaumatin-like
proteins (TLP). These pathogenesis related (PR) proteins are present in grape berries. In this
laboratory practical the proteins were extracted from the skin of the grape since it easily and
readily available throughout the different ripening stages of the grapes. The pulp was also
extracted and used for analysis. Specific proteins such as those responsible for photosynthesis
were exclusively found in the skin tissues. However, in both the pulp and skin tissues chitinases
and thaumatin-like proteins (TLPs) were identified. This quantification of chitinases and
thaumatin-like protein can be practical in tracing changes pathogenesis-related proteins during
the growth of grapes and during the ripening stages.

Practical Report on Proteomic Analysis of Grapes 3
Introduction
Abiotic stresses caused by global climate change, together with biotic stresses, negatively
affect crop growth and productivity throughout the world, resulting in a reduction of crop yields
by more than 50% Comprehensive analysis of grape proteome has to be conducted to show how
the networks in grape respond to external stress and the increasing tolerance of grape .The
grapevine is also scientifically referred to as Vitis vinifera L and it the world’s most important
crop. In the year 2007 FAO statistics show that the global annual grape production was around
sixty-five million tons and the land dedicated to grape farming was about 7.5 million hectares in
the world (Tian, 2015). The grape berries are consumed or they can be processed for purposes of
winemaking or must production. Nutraceutical and cosmetic businesses use extracts from grape
seeds, skin and leave to make various products. The seeds, skin, and leaves are also used for
research devotions.
The grape is known-climacteric fruit and it has double sigmoid growth pattern. Rapid
cell division, which leads to increase in the number of cells, and expansion of cells characterize
the first phase after fruit set. The second phase coincides with the beginning of ripening and it is
commonly referred to as veraison. Important physiological biochemical changes usually
characterize this phase and these changes include coloring and softening. Grapes change in
composition and size as they develop.

Practical Report on Proteomic Analysis of Grapes 4
The economic value of the berries and the quality of the wine produced depends on the
quality of the berries; therefore, the correct ripening is fundamental. This has led to research of
grape berry development through different approaches to determine the molecular level of the
changes undergone and the various factors that influence these changes. Gene by gene analysis
was among the first studies on development and ripening of grape berries. The availability of
vinifera and related species genomic resources as expressed sequence tags (ESTs) have enabled
mRNA expression profiling berry development studies in large scale.
Formation of protein haze is usually well thought-out as a wine fault and therefore wine
manufacturing companies must be very concerned of protein stabilization since the proteins in
wine can denature and lead to haze formation. The chief soluble proteins responsible for causing
haze formation are pathogen- related proteins that are available during the various stages of
grape berries ripening. This family of proteins was first discovered in tobacco due to the
oversensitive reaction of TMV (tobacco mosaic virus). They are acidic low molecular mass
molecules with relatively high proteolytic degradation under low pH values.
Most recently proteomic centered technologies have been applied successfully to analyze
the stress response and development of grape berries. The studies are mainly by means of single
two-dimensional gel electrophoresis (2-DE). Our understanding of the changes of various
proteins in grape berries development stages has been improved by the proteomic approach.
More robust quantitative proteomic methods such as mass spectrometry (MS), which is mainly,
centered on quantifiable steady isotopic labeling, increase our knowledge of grape berry
development. 2-DE is based on the difference in –gel electrophoresis (DIGE) it is as well a
robust technique that can be used to report the changing of several proteins during the ripening of
grape berries. The technique analyses samples using immunoblotting, liquid chromatography,

Practical Report on Proteomic Analysis of Grapes 5
and metabolic-stable isotope labeling. It is one of the most broadly applied strategies for
quantitative proteomic studies. For large volume spots, substantial comparative changes of
protein appearance can be measured.
This practical performed proteomic study on grape berries development and it was only
limited to the ripening period, from the onset of ripening to full ripening. The pericarp/mesocarp
tissues in grape berries during the various stages of ripening addressed the protein changes
expressed. The berries were sampled obtaining four biological duplicates to be analyzed for
proteome changes with the aid of 2-DE-based DIGE. Rigorous statistical analysis of the results
we obtained provides a global insight concerning the proteome changes taking place during the
development of grape berries. Comparing full ripened berries from different years will clearly
help detect inter-seasonal differences. For several decades the organic and sugar metabolism
during the various development stages of ripening grape berries have been characterized so well
to help us understand what molecular processes takes place. The main organic acid that is stored
in grape berry cells is malate and it accumulates the beginning of fruit formation all the way to
veraison .Imported carbon is the major source of malate synthesis but photosynthesis occurring
in the fruit at this stage may also contribute to synthesis of malate.
Materials and Methods
Material
The reagents involved in this experiment include 2-Mercaptoethanol, acetone, acid-
washed sand, trichloroacetic acid (TCA), thiourea, sucrose, SDS, sodium chloride, ready strips
IPG strips, protein assay dye reagent concentrate, protease inhibitor cocktail, PMSF, methanol,

Practical Report on Proteomic Analysis of Grapes 6
IPG buffer, ice, glycine, EDTA, glycerol, DTT, ammonium acetate,BSA standards ,
bromophenol blue, cetyltrimethylammonium bromide and CHAPS. 2-Mercaptoethanol, PMSF,
SDS, and methanol are very toxic when swallowed, inhaled or it gets into contact with skin,
therefore, caution should be taken when handling it. Acetone should be handled with care since it
is an unstable gas.
Development of the protocol
To facilitate crop proteomic analyses, our group has established sample preparation methods
that are suitable for 2-DE19,25–30 and studied proteomic changes in maize in response to drought
and heat stress29,31–33. We designed a novel protein extraction protocol that, for the first time,
integrates TCA/acetone precipi-tation with phenol extraction19,26. This protocol was originally
developed to extract proteins from the adult evergreen leaves of the olive tree (Olea europea L.),
which is an economically important oil-producing crop in many Mediterranean coun-tries. Olive
leaves contain large amounts of phenolic compounds (~15–70 mg g−1 fresh weight) primarily in
the form of oleu-ropein34. When olive leaves are destroyed by tissue disruption, oleuropein acts
as a very strong protein denaturant because it activates enzymes (e.g., β-glucosidase) in different
organelles35. This process may explain why the direct extraction of proteins from olive leaves in
aqueous extraction buffers followed by pro-tein precipitation results in brownish pellets that are
difficult to use in downstream processes. In the case of olive leaves, this integrated protocol is
more efficient than either TCA/acetone extraction or phenol extraction alone19.
Grape berries for that season were availed to us by our institution and they were allowed to ripen
naturally. Samples were continuously obtained from the berries during the development stages
from the start of ripening to full ripening. The first stage consisted of green berries. The second
phase which is considered the beginning of ripening was observed visually as the surface of the
berries started turning pink. The densities of these ripening berries were estimated through there
floatation in different solutions and this helped in classifying them. The berries were then sorted
according to their size and densities. The skin obtained from the berries during the different
ripening stages was used to conduct laboratory testing to determine the total acidity, color index,
and juice pH.
Protein extraction

Practical Report on Proteomic Analysis of Grapes 7
The grape berry skin (exocarp) was different during the different ripening stages. Before
protein is extracted from the skin of grape berries, the skin tissue was peeled away. Liquid
nitrogen was used to ground the exocarp tissues in a mortar and a fine powder was obtained.
Unless stated, each was followed by a twenty-minute centrifugation at 40C throughout the whole
procedure.
Twenty grams of grounded mesocarp tissue were homogenized in twenty millimeters of
the extraction that contained fifty mm of Na2HPO2, 1 mm EDTA, 0.1 mm ascorbic acid, one
percent polyvinylpyrrolidone (PVPP), one mm Na2O5S2, ten mm ascorbic acid and protease
inhibitors cocktail. The procedures were all carried out at temperatures of 40C. Filtration through
eight layers of cotton gauze was done on the homogenate and the filtrate centrifuged at 50000 g.
The pellet was washed away once in a buffer that contained fifty mm Na2HPO2 at pH 7, one mm
NaCl, ten mm ascorbic acid and the centrifuge was recovered at 35 000 g. The pellet was then
washed once with ethanol, twice with acetone that was 80 percent chilled. The recovered pellets
were dried at 40C and protein extraction was carried as described in the practical procedure.
The pellet was regulated in 400 μ phenol extraction buffer that was adjusted to pH 7.4
and protease inhibitors cocktail and it was incubated for thirty minutes under frequent vortexing.
Thiamin –saturated phenol at pH 7.5 was added at an equal volume. The mixture was then
incubated for approximately thirty minutes and vortex done after every five minutes.
Centrifugation for forty minutes at 14 500g resulted in a sharp phase separation. The upper phase
was successfully recovered and a second phenol extraction was done in the aqueous phase. The
recovered phenol was then precipitated with 0.1 mm ammonium acetate in methanol for a whole
night and the precipitate was again washed three times in 0.1 mm ammonium acetate followed
by two washings in eighty percent acetone.

Practical Report on Proteomic Analysis of Grapes 8
Labeling of proteins with CyDye
The proteins that had been precipitated and air-dried were solubilized in labeling buffer.
Protein concentration in the supernatant was quantified with the help of the RCDC technique
after the unsolvable material had been pelleted using centrifugation. 200 μg of protein was
modified to one microgram using labeling buffer and further cleaning with the Ettan 2D cleaning
–up kit was done in accordance with the manufacturer recommendations. The protein content
was measured again after the precipitated protein had been solubilized in thirty microliters of
labeling buffer. Labeling with Cy2 was done using internal standards for a mixture containing
equal amounts of protein.
2-DE and image scanning
20 μl each of Cy3, Cy5, and Cy2 labeled sample was combined with 60 microliters of
sample buffer one before the mixture was incubated for ten minutes in ice to enable 2-D DIGE
analysis (Li, 2016). The samples were then adjusted to 150 microliters of the sample buffer 2 and
they were submitted as the first dimension separation to IEF. IPG strips were swollen overnight
and a cup-loading method was used to apply the samples. Two steps were focused and two strips
were acquired as a result. A Typhoon 9410 scanner enabled images of Cy3, Cy2, and Cy5 to be
acquired according to the recommendations of the manufacturer.
DIGE image analysis
Progenesis Same Spots v3.0 software was used to analyze the DIGE images obtained in
the practical procedure. Alignment of the images was performed and prominent spots were used
to assign 60 vectors manually and images were digitized within each gel. Additional vector were
added using the automatic vector tool and then the vectors were revised and edited for correction

Practical Report on Proteomic Analysis of Grapes 9
manually. The gel images were aligned using the vectors according to a reference image of one
international standard (Hopper, 2013). Spots were revised manually with editing methods after
automatic detection and gel groups successfully established in accordance with experimental
design. Statistical significant differentiated spots analyzed during the different development
stages of grape berries were selected using spot-normalized volume technique. The selected
sports were analyzed and grouped regarding their copiousness pattern and they were clustered
hierarchically. Principle component scrutiny applied in progenesis same spots was used to assess
the spots.
Spot picking and tryptic digestion
Sports picking was done on 2-D gels that were loaded with 1mf of protein. The gels were
stained with Coomassie blue method and then scanning through a transmission-light
densitometer that was aligned with the DGE reference image. A manual spot picker was used to
remove the spots. Trypsin in –gel digestion was conducted and the dye and SDS impurities were
removed by extensive washing of the gel.
MS protein identification
The tryptic fragments were first desalted by means of Zip Tips according instruction from
the instructor for analysis in the TOF instrument. Eluted peptides were dehydrated completely in
a vacuum centrifuge and then they were resuspended in triflouroacetic acid. The 4700 proteomic
analyzer helped us to acquire the MS and MS/MS statistics.
RESULTS
Numerous proteins that are generally related to salt, oxidative and stress were
successfully identified through the ripening the various stages of the grape berries. Ascorbate,
glutathione, polyphenol oxidase, catalase, thioredoxin-dependent peroxidases, and superoxide

Practical Report on Proteomic Analysis of Grapes 10
were some of the enzymes that were detected to influence oxidative stress. The 2-DE proteomic
study shows that these are more highly expressed in the green tissue compared to tissues of ripe
grape berries. Ascorbate peroxidase and catalase are general detoxifier enzymes of ROS
(reactive oxygen species) while peroxiredoxins have a specific role in detoxifying reactive
oxygen species in chloroplasts. Ascorbates peroxidase decrease continuously after veraison. This
shows how stress plays a development role in the ripening of the grape berries.
Analysis of Results
A plasmid is a physically separated DNA molecule within the tissue and it affects the
proteome of by independent replication of itself. I think this greatly affects the accumulation of
fructose and glucose in the tissues of ripening fruit (Tian, 2015). The independent replication of
plasmids leads to variations in the proteins detected during the various ripening stages of the
grape barriers. At veraison, speedy proliferation in the mRNA levels of cDNAs occurs and this is
not observed in unripe grapes (Wang, 2017). These clones had homologs that are involved in the
structure of the cell.
The data and facts from other students are easily producible since the practical was done
on similar species of grapes and under the same environmental process. As long as the other
students followed the experimental procedure as given by the instructor then the results are
expected to be absolutely similar (Sarry, 2010). Small variations may just come up due to
measurements and observing techniques but they should not be significant enough to alter the
facts in the practical.
These results precisely provide an evidence of a strong relationship between ripening of
the grape berries and the accumulation of pathogenic related proteins (Wang, 2017). Factors such
as osmotic stress caused by high free hexoses level induce the accumulation of some pathogenic

Practical Report on Proteomic Analysis of Grapes 11
related proteins during ripening. Salicylic acid, acetic acid, and ABA which are signaling
molecules play a significant role in influencing the accumulation of pathogen-related proteins.
Therefore the accumulation of PR proteins is clearly triggered by endogenous influences.
Several isoforms of the cyclase protein family were identified both in the green and ripe berries.
They belong to the diterpene analyses family according to the phylogenetic and multi-alignment
analysis techniques (Tian, 2015). The possible role of GA in the grape berry development could
be enlargement of the berries. The proteomic results coincide with the theoretical facts that we
learned in the theory part of the topic.
Conclusion
The detection of proteome changes that take place during the different successive stages
of grape berries development has been possible by the application of DIGE techniques. Pre-
veraison and after veraison are the two key points detected in this practical (Wang, 2017). There
is a change in from the net accumulation to degradation of malate just before veraison occurs. In
pre-veraison phase; there were intense changes in the amount and level of proteins structured
while during the after-veraison phase relatively minor changes were observed in the grape
berries (Giribaidi, 2010). The proteins detected in the practical belonged to a broad array of
metabolic developments. The proteomic analysis of grape cells during ripening is not conclusive
therefore research on this area is still underway. The was time-consuming and involve rigorous
analysis of data obtained so as to come up with useful information to improve our understanding
of proteomic in ripening grape barriers as well as other related fruits.
To make the data obtained in such a practical, I think more techniques should be used in
approaching it. This would enable comparison of the data obtained using the various techniques.
Furthermore, more precise analysis methods should be applied to effectively interpret the data.

Practical Report on Proteomic Analysis of Grapes 12
Despite the challenges experienced in the practical, we can conclude that the experiment was
successful since the intended objectives were met.
Bibliography
Burton, H. (2016). Grape skin extract induces an Unfolded Protein Response. New Delhi: Shamir
Publishers.
Fennel, G. a. (2015). Protein identificatio and quantification from riverbank grapes. London: Sir Jackson
Publishers.
George, P. a. (2015). Quantitative proteomic analysis of cabinet sauvignon grape cells exposed to
thermal stresses. Chicago: Long Horn Publishers.
Giribaidi, G. a. (2010). Proteomic analysis of the effects of ABA treatment on ripening Vitis vinifera
berries. New York: Springer Publication.
Hopper, C. a. (2013). Protein analysis indicate massive changes in metabolism prior to growth and
photosynthesis of grapevine. New York: New York Press.
Li, W. a. (2016). Heat acclimation reduces oostharvest loss of table grapes during cold storage. Chicago:
Flamingo Works.
Sarry, S. a. (2010). Grape berry biochemistry revisited upon proteomic analysis of the mesocarp.
Singerpore: Adventure Press Works.
Tian, H. &. (2015). Proteomic analysis of Sauvignon Blanc grape skin,pulp and seed and relative
quantification of pathogenesis-related proteins. Chicago: Springer Publishers.
Wang, D. a. (2015). Differential proteomic analysis of grapevine leaves. New York: ANC publishers.
Wang, W. a. (2017). System- level and granger network analysis of integrated proteomic and metabolic
dynamic identifies key points of grape berry development at the interface of prmary and
secondary metabolism. Ney York: New York Press .
Giribaldi, M., Gény, L., Delrot, S. and Schubert, A., 2010. Proteomic analysis of the effects of ABA treatments on
ripening Vitis vinifera berries. Journal of Experimental Botany, 61(9), pp.2447-2458.