Establishing a Leaf Proteome Reference Map for Ginkgo biloba

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Article

Establishing a leaf proteome reference map for Ginkgo biloba provides insight into potential ethnobotanical uses Lubica Uvackova, Emilia Ondruskova, Maksym Danchenko, Ludovit Skultety, Ján A. Miernyk, Pavel Hrubik, and Martin Hajduch J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf503375a • Publication Date (Web): 03 Nov 2014 Downloaded from http://pubs.acs.org on November 8, 2014

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Journal of Agricultural and Food Chemistry

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Title:

Establishing a leaf proteome reference map for Ginkgo biloba provides insight into potential ethnobotanical uses

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Authors:

Lubica Uvackova1, Emilia Ondruskova2, Maksym Danchenko3, Ludovit Skultety3,4, Ján A. Miernyk5,6, Pavel Hrubík7, Martin Hajduch1,3

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Affiliations:

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Department of Reproduction and Developmental Biology, Institute of Plant

Genetics and Biotechnology, Slovak Academy of Sciences, Nitra, Slovakia;

9 10

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Academy of Sciences, Nitra, Slovakia; 3Institute of Virology, Slovak Academy

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of Sciences, Bratislava, Slovakia; 4Institute of Microbiology, Academy of

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Sciences of Czech republic, Prague, Czech Republic; 5USDA, Agricultural

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Research Service, Plant Genetics Research Unit; 6Division of Biochemistry,

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University of Missouri, Columbia, USA; 7Faculty of Horticulture and Landscape

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Engineering, Slovak University of Agriculture, Nitra, Slovakia.

Department of Woody Plant Biology, Institute of Forest Ecology, Slovak

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Corresponding Author:

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Martin Hajduch, Department of Reproduction and Developmental Biology,

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Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences,

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Nitra, Slovakia; Telephone, 421-37-6943346; Fax, 421-37-7336660; E-mail,

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[email protected]

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Keywords:

Maidenhair tree, king cobra, medicinal tree, ornamental tree, 2-DE, protein map

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Abstract

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Although ginkgo (Maidenhair tree, Ginkgo biloba L.) is an ancient medicinal and ornamental

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tree, there has not previously been any systematic proteomic study of the leaves. Herein we

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describe results from the initial study identifying abundant ginkgo leaf proteins and present a

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gel reference map. Proteins were isolated from fully developed mature leaves in biological

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triplicate and analyzed by two-dimensional electrophoresis plus tandem mass spectrometry.

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Using this approach we were able to reproducibly quantify 190 abundant protein spots, from

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which 158 proteins were identified. Most of identified proteins are associated with the energy

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and protein destination / storage categories. The reference map provides a basis for

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understanding the accumulation of flavonoids and other phenolic compounds in mature

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leaves (e.g., identification of chalcone synthase, the first committed enzyme in flavonoid

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biosynthesis). We additionally detected several proteins of as yet unknown function. These

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proteins comprise a pool of potential targets that might be useful in non-traditional medical

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applications.

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Introduction

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Ginkgo (Ginkgo biloba L.) or maidenhair tree is a “living fossil” among plants and is the only

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surviving representative of the family Ginkgoaceae (1-3). Ginkgo is an important medicinal

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and ornamental tree that is largely resistant to disease and pests (4) and inhabits diverse

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environments. The nuts and the leaves of this tree contain a unique cohort of chemicals that

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have been used for five thousand years in traditional Chinese medicine (5, 6), and ginkgo

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remains a popular medicinal plant today. Leaf extracts contain secondary metabolites, such

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as terpene trilactones (ginkgolides and bilobalide) and flavonol glycosides, that have been

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suggested to improve memory. Furthermore, ginkgo leaf extracts have been used to

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increase peripheral and cerebral blood flow (7), in the therapy of cognitive decline, and to

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treat Alzheimer’s disease (8, 9). Finally, Ginkgo leaves have also been used in alternative

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anticancer treatments (10, 11).

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Proteomic analyses of trees are complicated by the lack of well-developed database

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resources. Despite this, descriptions of several angiosperm leaf proteomes have been

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published (12), including teak (Tectoma grandis; 13), spruce (Picea 14,15), poplar (Populus;

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16, 17), and oak (Quercus; 18). Additionally, the analysis of gymnosperm “leaves” (Pinus

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massoniana needles) led to the identification of 95 proteins, separated into eight functional

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groups, that changed in abundance in response to calcium nutrition (19).

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One of the few studies related to the ginkgo proteome provided new insights into the

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process of ploidy in male gametes and the authors noted that colchicine treatment of

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microsporocytes resulted in a low mutation rate of diploid male gametes (20). Another study

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relevant to the ginkgo leaf proteome provides evidence that chloroplast senescence-related

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proteins were regulated at the protein rather than the transcript level, helping to understand

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senescence regulation in woody species (21). Herein, we describe results from the first

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systematic investigation of the abundant ginkgo leaf proteins.

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Material and Methods

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Plant material



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Fully mature leaves of a single male Maidenhair tree (Ginkgo biloba L.) growing in the City

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Park, Nitra (Slovakia) were harvested from the ends of branches on a sunny morning, and

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stored at -80°C until analyzed. For the analysis, 15 leaves were combined and homogenized

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together, representing one biological replicate. In total, three biological replicates were

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analyzed in this study.

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Protein Extraction

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Proteins were extracted using a method previously described (22). One gram fresh weight of

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leaves was ground to a fine powder with a mortar and pestle containing liquid N2. Proteins

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were extracted by shaking the leaf powder in a solution containing 50% (v/v) phenol, 0.45M

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sucrose, 5mM EDTA, 0.2% (v/v) 2-mercaptoethanol, and 50 mM Tris-HCl, pH 8.8 for 30 min

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at 4°C. After centrifugation at 5000g for 10 min at 4°C the phenol phase was transferred to

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clean tube and proteins were precipitated by adding 5 volumes of ice-cold 0.1M ammonium

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bicarbonate (ABC) in methanol followed by overnight incubation at -20°C. After

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centrifugation at 5,000 x g and 4°C for 10 min, protein pellets were washed twice with ice-

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cold 0.1 M ammonium acetate in methanol, twice with ice-cold 80% acetone, and once in ice-

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cold 70% ethanol.

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Two-dimensional gel electrophoresis and “in-gel” digestion

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Proteins were dissolved in rehydration sample buffer (8M urea, 2M thiourea, 2% (w/v)

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CHAPS, 2% (w/v) Triton X-100, 50mM DTT, 0.5% ampholytes pH 3-10), and protein

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concentrations were determined according to Bradford (23). Then, 50 µg of proteins were

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analyzed by 2-dimensional electrophoresis (2DE) using 7 cm immobilized pH gradient (IPG)

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strips with pH range 5-8, as described earlier (24). Briefly, isoelectric focusing (IEF) was

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performed using a Protean IEF Cell (Bio-Rad) programmed for active rehydration for 12

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hours at 50 V followed by 150 V for 150 VH, 500 V for 500 VH, and 4000 V for 15000 VH.

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After IEF, the IPG strips were equilibrated in 6 M urea containing 30% (v/v) glycerol, 70 mM

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SDS, and 0.006% (w/v) bromphenol blue) for 15 min in equilibration solution with 1% (w/v)

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DTT, then for an additional 15 min in equilibration solution containing 2.5% (w/v)

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iodoacetamide. Equilibrated IPG strips were placed on top of 10% SDS-gels and 4 ACS Paragon Plus Environment

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electrophoretic separation was performed at 10mA per gel. Finally, triplicate gels were

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stained with colloidal Coomassie blue, the images digitalized (GS-800, Bio-Rad, Hercules,

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CA) at 300 dpi / 16 bit grayscale and quantified using PDQuest 8.0 software (Supplemental

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Figure 1).

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Protein spots present in at least in two of the triplicate gels were excised, de-stained

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in wash solution (50% acetonitrile (ACN), 50mM ABC), and dehydrated in 100% ACN. The

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dehydrated gel plugs were rehydrated in 50 mM ABC containing modified sequencing grade

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trypsin (Promega), and digested at 37°C overnight. Tryptic peptides were extracted with

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60% ACN containing 1% formic acid (FA). Solutions were reduced to dryness using a

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Concentrator plus (Eppendorf, Germany). The tryptic peptides were dissolved in 2% ACN

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containing 0.1% FA, and stored at -80ºC until used.

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Protein identification by liquid chromatography-tandem mass spectrometry

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In total, 5 µL of tryptic peptides were separated using a nanoAcquity UPLC system (Waters,

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USA). Samples were injected onto a Symmetry C18 trap column (20 mm length, 180 µm

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diameter, 5 µm particle size). After 3 min of desalting/concentration with 3% ACN containing

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0.1% FA at a flow rate 10 µl·min-1, peptides were separated on an analytical BEH130 C18

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column (150 mm length, 75 µm diameter, 1.7 µm particle size). For quick profiling, a 15 min

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gradient of 6–40% ACN containing 0.1% FA at a flow rate of 350 nl·min-1 was applied. The

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column outlet was connected to a precut non-coated PicoTip emitter (360 µm outer diameter,

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20 µm inner diameter, 10 µm tip diameter; New Objective, USA); mounted into the nanospray

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source of a Q-TOF Premier tandem mass spectrometer (Waters, USA). The instrument was

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controlled by MassLynx software v. 4.1 (Waters, USA). Basic settings were: source

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temperature, 70°C; nanospray voltage, 3.4 kV; nebulizer gas pressure, 0.4 bar; collision gas

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flow, 0.48 ml·min-1. Data were acquired using the MSE method, which performed alternate

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scans at low and high collision energies. The quadrupole analyzer was operated in the

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radio-frequency mode, allowing all ions to reach the TOF analyzer, so that in a single full-

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scan run both precursor ions and accurate fragment masses were detected. In the MS

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channel, data were collected at a constant collision energy of 4 eV while in the tandem 5 ACS Paragon Plus Environment


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(MS/MS) channel fragments were recorded while the collision energy was ramped up from

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20 to 35 eV. Spectra acquisition scan rate was 0.8 s, with a 0.05 s inter-scan delay, ions

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with 100-1900 m·z-1 were detected in both channels. The quadrupole mass profile settings

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allowed efficient deflection of masses of less than 400 in the low energy mode enabling

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filtering of contaminating ions.

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Analysis of the MS/MS data

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Peak smoothing was performed, including background-subtraction, centroiding, and

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deisotoping. Noise filtering thresholds where set at: intensity, 1200; low energy, 150; high

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energy, 20 counts. All data were lock-spray calibrated against Glu-Fibrinopeptide B, using

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data collected from the reference line during acquisition (500 fmol·µl-1 in 25% ACN with 0.1%

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FA, flow rate 350 nl·min-1, scans every 30 s). The lockmass-corrected data were

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deconvoluted to produce a single accurate monoisotopic mass for each peptide and

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associated fragment ions. Differently-charged precursors were summed. The initial

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correlation of precursor and fragment ions was achieved by means of time alignment.

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The MS/MS data were processed by the ProteinLynx Global Server (PLGS) v. 2.4

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(Waters, USA) against an in-house database consisting of sequences downloaded from

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UniProt during July 2013. In total, 742 Ginkgo species entries, 856,836 Spermatophyta

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UniRef90 databank entries (taxonomy filter Spermatophyta and 90% sequence redundancy

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grouping), and 1,573,198 Viridiplantae entries were included. The databank was randomized

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to allow efficient correction for false-positive matches. The search parameters used were:

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fixed modification, carbamidomethyl-Cys; variable modification, oxidized Met; one missed

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cleavage site; at least 7 fragment ions per protein, and an automatic error tolerance value.

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Protein identifications were accepted after manual inspection of probabilistic PLGS

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assignment at the 95% confidence level. Additional parameters to accept protein

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identification were; at least two peptides matched to the protein sequence, a minimum of

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three fragment ions per peptide, a 50 ppm tolerance of database-generated theoretical

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peptide ion masses, and a PLGS score greater than 50. The PLGS score is a statistical


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measure of accuracy of assignation and was calculated using a Monte Carlo algorithm. A

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higher score indicates a greater confidence in the assigned protein identity.

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Results

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Two-DE combined with MS/MS was used for analysis of the abundant gingko leaf proteins

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(Fig 1). Using this approach we reliably quantified 190 abundant 2-DE spots (Supplemental

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table 1); of which 158 were identified by MS/MS (Table 1). These data were used to

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establish the initial ginkgo leaf proteome reference map (Fig. 2). As expected, the most

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abundant 2-DE spots were identified as ribulose-1,5-bisphosphate carboxylase/oxygenase

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(RuBisCO) subunits (spots 5502, 6509, 4504, 6501, and 5501). The next most abundant

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proteins were ATP synthase (spot 1506), RuBisCO activase (spot 2304), and the Class III

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homeodomain leucine-zipper protein C3HDZ3 (spot 2401) (Fig. 2). The 2-DE spots with the

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lowest abundance values that contained an identifiable protein were spot 2708 (ginancin),

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followed by spot 8401 (alanine aminotransferase), spot 5105 (superoxide dismutase), and

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spot 6307 (protein similar to glycerate dehydrogenase/hyrdoxypyruvate reductase.

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Most of the identified ginkgo leaf proteins were assigned to the energy group

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Identified proteins presented on the reference map were assigned to one of 10 functional

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groups (Fig. 3) according to Bevan et al. (25). Only one primary function was assigned per

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protein. Most of the identified proteins were associated with the energy group (61). Within

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this group, 24 2-DE spots were identified as subunits of RuBisCO. In total, 10 2-DE spots

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were identified as ATP synthase subunits (Table 1), 4 spots were identified as

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fructosebisphosphate aldolase, and another 4 as phosphoglycerate-kinase.

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The second most populated category is proteins associated with destination/storage

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(31). Within this group, 14 2-DE spots were identified as Ginnacin, the 11S ginkgo seed

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storage protein (26). Four 2-DE spots were identified as subunits of the ATP dependent Clp

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protease (Table 1). A total of 16 proteins associated with disease/defense comprise the third

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largest functional group (Table 1). Five 2-DE spots contained proteins associated with

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abscisic acid stress and ripening (ASR) (27). The fourth largest functional group was

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proteins of unknown function (PUF; 15). 7 ACS Paragon Plus Environment


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The relative volumes of all proteins within a particular functional class were summed

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in order to determine the most abundant functional group (Supplemental Figure 2). Proteins

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associated with energy contributed more than 70% to total protein abundance, followed by

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those associated with destination / storage, and disease / defense. The fourth most

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abundant functional group was proteins associated with transcription, followed by PUF

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(Supplemental Figure 2).

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An abundant protein of unknown function is similar to nucleolar protein 58 of the king

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cobra

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We detected 15 proteins of unknown function (PUF). These proteins were subjected to

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BLASTP searches in order to reveal similarity with known proteins (Supplemental table 2).

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The most abundant protein in this group was Os01g0217100 (spots 1201, 3403, and 7301)

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with UniProt accession number Q5QNF4 (Table 1). The NCBI Blast search revealed that the

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amino acid sequence of this unknown protein is 53% similar to the amino acid sequence of a

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purine permease from Foxtail millet (Setaria italica) (Supplemental table 2). Unexpectedly,

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the second most abundant PUF was a putative uncharacterized protein (G7KFS4, spot 1501)

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that has the closest similarity (56%) to nucleolar protein 58 from the king cobra

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(Ophiophagus hannah) (Supplemental table 2). Third most abundant PUF was

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uncharacterized protein with UniProt accession M0TUE2 (Table 1). A BLAST search failed

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to reveal similarity to any known protein (Supplemental table 2).

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Discussion

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The aim of this study was to use 2-DE based proteomics approach to establish a reference

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map of most abundant ginkgo leaf proteins. The application of a proteomics strategy for the

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characterization of medicinal plants is not new (e.g., studies of Catharanthus roseus (28),

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Pinellia ternata (29), Podophyllum hexandrum (30), and Indian ginseng (31)). The previous

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analyses yielded meaningful results, such as in the case of Gynura procumbens where the

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92 characterized proteins from leaves included miraculin, a taste-masking agent with high

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commercial value (32).


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The majority of the proteins presented on the reference map (Fig. 2) are involved with

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photosynthesis and energy metabolism. The abundance of many of these proteins in leaf

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proteomes has been noted previously (e.g., teak (13), Indian ginseng (31), Gynura

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procumbens (32), barrel medic (33), tomato (34), holm oak (18), and Miscanthum sinensis

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(35)). In the present study we detected multiple spots containing subunits of RuBisCO (spots

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5502, 6509, 4504, 6501, and 5501), the enzyme that catalyzes the first step of carbon

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fixation in Calvin cycle, and the most abundant protein in the Ginkgo leaf proteome.

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Chloroplastidial ATP synthase, which generates ATP using a proton gradient, was

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represented by 10 2-DE spots on the reference map and was the second most abundant leaf

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protein (Fig. 2). Half of the ATP synthase spots were identified as the α−subunit, and the

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other five as the β−subunit (Table 1).

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Glycolytic enzymes are well represented on the 2-DE reference map. Four spots

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were identified as fructose-bisphosphate aldolase (FBA), which catalyzes the aldol cleavage

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of fructose-1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone-

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phosphate. Another three 2-DE spots (6407, 6403, 7302) were identified as glyceraldehyde

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3 phosphate dehydrogenase, which catalyzes the conversion of glyceraldehyde 3 phosphate

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to D-glycerate-1,3-bisphosphate. Two 2-DE spots (5302, 6305) were identified as the Krebs

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cycle enzyme NAD-malate dehydrogenase.

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Several stress-related proteins are present on the 2-DE map, including ascorbate

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peroxidase (APX, spots 5204 and 6104) and superoxide dismutase (SOD, spots 3101, 5105

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and 7105). The APX reaction is part of the ascorbate-glutathione cycle for protection against

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oxidative stress. It uses two molecules of ascorbate to reduce H2O2 to water (36). The SOD

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reaction is also involved with H2O2 detoxification (37).

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Proteins associated with secondary metabolism and proteins of unknown function are

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abundant in the ginkgo leaf proteome

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Many of the spots on the reference map correspond to enzymes involved in secondary

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metabolism. Secondary metabolites are valuable targets for industrial applications (38, 39).

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It is known that Ginkgo´s leaves contain biologically active terpenes and a range of flavonoid 9 ACS Paragon Plus Environment


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glycosides (40). More than 38 leaf flavonoids have been isolated so far (41). Elaboration of

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the flavone core structure yields structurally related compounds with diverse functions (42,

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43). In the present study we detected 5 proteins associated with flavonoid metabolism;

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caffeoy-CoA O-methyltransferase (spot 1206), cinnamoyl-CoA reductase (spot 303),

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anthocyanidin reductase (spot 4306), an isoflavone reductase-like protein (spot 6204), and

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chalcone synthase (spot 6405). Chalcone synthase catalyzes the first committed step in

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flavonoid biosynthesis (44), producing chalcone from which all other flavonoids are derived.

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Anthocyanidin reductase (spot 4306) converts anthocyanidins to their corresponding 2,3-cis-

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flavan-3-ols, and is responsible for formation of epicatechin and epigallocatechin in

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Arabidopsis and Medicago (45). Flavonoids comprise a diverse group of polyphenolic

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compounds with neuroprotective properties in models of Alzheimer's disease, stroke, and

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Parkinson's disease (46). Isoflavone reductase reduces achiral isoflavones to chiral

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isoflavanones in Ginkgo (47). Cinnamoyl-CoA reductase (CCR) and caffeoyl-CoA O-

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methyltransferase (CCoAOMT) are enzymes active in phenylpropanoid biosynthesis. The

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CCR-catalyzed reaction is the first step of the lignin-specific branch of the phenylpropanoid

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pathway and is a key enzyme responsible for quality and quantity of lignin (48-50).

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Proteins with unknown function (PUF) are fifth most abundant functional group on

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established map of ginkgo leaf proteome (Fig. 2). Most abundant PUF was Os01g0217100

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protein from rice (spot s1201, 3403, and 7301) with UniProt accession number Q5QNF4

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(Table 1). Using the BLAST algorithm to search the NCBI databases revealed that the amino

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acid sequence of this protein is 77% similar to rice amino acid transporter-like protein

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(Supplemental table 2). The nucleotide sequence for this protein was described during the

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sequencing of rice genome (51).

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The second most abundant PUF was a putative uncharacterized protein (G7KFS4)

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that is for 56% similar to nucleolar protein 58 characterized from the blood of the king cobra

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(Ophiophagus hannah) (52) (Supplemental table 2). Third most abundant PUF protein was

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uncharacterized protein (spot 5101, M0TUE2). This protein showed similarity only to other

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unknown proteins (Supplemental table 2). Additionally, this study quantified 10 other 10 ACS Paragon Plus Environment

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unknown proteins from ginkgo leaves (Table 1). It should be noted that unknown proteins

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constitute more than half of the proteins in plant genomes (53). The characterization of

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unknown proteins is one of the greatest challenges of post-genomics biology, and should

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lead to a broader understanding of metabolic processes. The ginkgo leaf PUF’s constitute a

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pool of targets related to traditional medicine-related applications (Fig. 4).

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Conclusions

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A quantitative proteomics approach to analysis of the ginkgo leaf proteome led to detection

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of several proteins associated with secondary metabolism (Fig. 4). Additionally, we also

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detected 15 proteins of unknown function that constitute pool of potential targets for

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ethnobotanical applications (Fig. 4). Follow-up studies will be required to further characterize

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the ginkgo leaf PUF’s and their potential contributions to the health benefits alluded to for

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centuries in traditional Chinese medicine.

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Acknowledgements

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This research was partially funded by the European Community under project number

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26220220180: Building Research Centre „AgroBioTech" and by the Research &

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Development Operational Program funded by the ERDF - Centre of Excellence for White-

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Green Biotechnology (ITMS 26220120054).

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Figure legends

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Figure 1. The experimental workflow. Proteins were isolated from ginkgo leaves using a

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phenol-based extraction method and separated by two-dimensional electrophoresis. Protein

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spots were quantified using the PDQuest software. Excised spots were digested with

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trypsin, separated by nanoAcquity UPLC, and analyzed by MS/MS in the MSE mode.

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Proteins were identified using ProteinLynx Global Server.

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Figure 2. Map positions of 158 proteins extracted from Ginkgo biloba leaves. This

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quantitative reference map was established using 7 cm IPG strips (pI 5-8), PDQuest software

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(version 8.0), and Q-TOF Premier mass spectrometer.

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Figure 3. Functional classification of 158 proteins identified from Ginkgo biloba leaves,

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based upon the method of Bevan et al. (25).

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Figure 4. Quantitative analysis of proteins associated with synthesis of phenolic and

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flavonoids compounds with ethnobotanical applications. This approach was also applied to

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analysis of proteins of unknown function (PUF) that are potential targets for further analysis

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and with potential use in non-traditional medical applications.

299 300

Supplemental material

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Supplemental Figure 1. Two-DE gels of the Ginkgo leaf proteome, presented in biological

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triplicate.

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Supplemental Figure 2. The summed relative volumes (%V) for all proteins assigned to

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each functional group.

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Supplemental Table 1. Relative volumes (%V) of all quantified 2-DE spots presented on 7

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cm 2-DE gels of pH 5-8.


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Supplemental Table 2. The results of BLASTP queries of Proteins of Unknown Functions

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(PUF) against NCBInr protein database, sorted by percent identity (Max ident). The top

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three matches are shown.

310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325



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Table 1. Proteins located on the Ginkgo biloba leaf reference map. The table includes 2-DE

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spot number, UniProt accession number; description / name, observed MW (kDa) / pI,

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deduced MW (kDa) / pI, PLGS (Protein Lynx Global Server) score, number of peptides

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matched to protein sequence, and relative volume of 2-DE spot ± SD (%V±STDEV) of

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biological triplicate analyses (Supplemental table 1).

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Spot No. UniProt Description 01 Metabolism 01.01 Amino Acids 3409 H8YHW0 Adenosylmethionine synthetase 3401 H8YHW0 Adenosylmethionine synthetase 3406 H8YHW0 Adenosylmethionine synthetase 8401 Q1M301 Alanine aminotransferase 7209 A9NUJ0 Aspartate aminotransferase 4402 M4CBV6 Glutamine synthetase 6307 I1NWA3 Glycerate dehydrogenase 6704 Q2YHN6 Methionine synthase 4703 B0ZQ27 Phenylalanine ammonia lyase 01.03 Nucleotides 4303 B9T0A9 Adenosine kinase putative 01.05 Sugars and polysaccharides 2504 B8LNV7 Glucose 1 phosphate adenylyltransferase 4201 B9T2C3 UDP glucosyltransferase putative 01.06 Lipid and sterol 5701 H9A985 Acetyl coenzyme A 7801 H9A985 Acetyl coenzyme A 02 Energy 02.01 Glycolysis 4409 I1KTX8 Phosphoglycerate kinase 3413 O24330 Phosphoglycerate kinase 4403 I1KTX8 Phosphoglycerate kinase 5307 M7ZR60 Phosphoglycerate kinase 02.02 Gluconeogenesis 6305 G3BMW0 Malate dehydrogenase 5302 O48906 Malate dehydrogenase 02.07 Pentose phosphate 7102 A9NP17 Ribulose-phosphate 3-epimerase 2303 C4PAW8 Sedoheptulose 1 7 bisphosphatase 5703 A5AS94 Transketolase 5702 I1H1V5 Transketolase 02.01 Glycolysis 7202 A9NUC9 Fructose bisphosphate aldolase 6303 Q8LK59 Fructose bisphosphate aldolase 6316 I3SDW4 Fructose bisphosphate aldolase 6310 B9SJY9 Fructose bisphosphate aldolase 6403 B9TQJ4 Glyceraldehyde 3 phosphate dehydrogenase 6407 P12859 Glyceraldehyde 3 phosphate dehydrogenase B 7302 P09315 Glyceraldehyde-3-phosphate dehydrogenase A 2206 A9NRN6 Triosephosphate isomerase

Exp. Theoret. MW/pI MW/pI (kDa) (kDa)

PLGS Score

Pep %V±STDEV

45/6.24 49/6.03 47/6.22 50/7.82 23/7.38 40/6.42 40/7.1 105/7.1 84/6.63

16/6.04 16/6.04 16/6.04 21/5.19 48/6.99 39/7.91 44/6.37 18/5.83 79/5.91

143.12 1575.12 1147.95 445.85 421.16 325.49 499.95 461.24 59.79

2 3 6 2 5 2 6 6 2

0.28±0.06 0.10±0.02 0.10±0.09 0.01±0.00 0.23±0.11 0.28±0.02 0.03±0.03 0.16±0.06 0.42±0.28

37/6.5

38/4.99

221.05

3

0.06±0.04

55/5.8 29/6.3

58/5.94 54/5.68

230.2 68.26

9 2

0.10±0.01 0.07±0.02

90/6.8 36/6.9 110/7.5 36/6.9

58.46 134.48

3 3

0.20±0.08 0.13±0.02

44/6.6 41/6.28 46/6.4 43/6.6

50/8.73 24/6.53 50/8.73 49/5.26

2168.39 84.99 869.69 730.62

12 3 8 5

0.46±0.18 0.28±0.08 0.19±0.08 0.17±0.11

32/7.1 84/6.6

35/9.22 43/7.99

2004.64 512.49

6 8

0.18±0.02 0.10±0.01

15/7.5 39/5.7 91/6.9 80/6.8

31/7.93 42/5.89 74/6.37 80/5.9

594,00 4 257.02 10 220.84 4 130.77 3

0.09±0.06 0.41±0.06 0.22±0.12 0.09±0.00

29/7.65 35/7.08 30/6.9 28/7.32 41/7.12 41/7.4 35/7.6 19/5.63

43/8.07 34/9.53 43/8.2 43/8.58 21/5.64 48/7.53 44/8.26 27/4.82

2368.93 267.41 135.71 228.15 866.6 4794.78 799.35 1349.52

1.14±0.33 0.18±0.09 0.09±0.02 0.07±0.05 0.35±0.05 0.43±0.21 0.41±0.06 0.06±0.04


10 4 3 4 3 19 9 5

Page 15 of 25

332


02.13 Respiration 602 I6N9D1 ATP synthase subunit alpha 1605 Q4FGJ3 ATP synthase subunit alpha 1611 I6N9D1 ATP synthase subunit alpha 2618 H7CG64 ATP synthase subunit alpha 1609 Q4FGJ3 ATP synthase subunit alpha 1507 Q9MRI7 ATP synthase subunit beta 1506 H9A983 ATP synthase subunit beta 1607 H9A983 ATP synthase subunit beta 2601 Q9MRI7 ATP synthase subunit beta 2603 Q9MRI7 ATP synthase subunit beta 02.30 Photosynthesis 7203 D8QZW2 Carbonic anhydrase 5201 A9NNY0 Carbonic anhydrase 2304 F8UAA7 Chloroplast rubisco activase 7804 G4XWZ6 Glycine decarboxylase complex subunit P 1204 Q39768 LHCB 1110 Q39768 LHCB 2211 O49079 Oxygen evolving enhancer protein 1 3211 O49079 Oxygen evolving enhancer protein 1 3404 B1PIE1 RuBisCo oxygenase activase alpha 2 2307 B1PIE1 RuBisCo oxygenase activase alpha 2 8502 I6N9F4 RuBisCo large chain 5303 I2AP09 RuBisCo large chain 6601 P48704 RuBisCo large chain 3501 I2AP09 RuBisCo large chain 5706 P48704 RuBisCo large chain 5912 E7E875 RuBisCo large chain 3818 P48704 RuBisCo large chain 5502 I6N9F4 RuBisCo large chain 6509 I2AP09 RuBisCo large chain 4504 I6N9F4 RuBisCo large chain 5501 I6N9F4 RuBisCo large chain 4605 I6N9F4 RuBisCo large chain 6814 M8A2T1 RuBisCo large chain 2616 K9LMB1 RuBisCo large chain 6501 J7QBH1 RuBisCo large chain 6506 E7E875 RuBisCo large chain 4602 H8YV26 RuBisCo large chain 6508 H8YV26 RuBisCo large chain 5907 H8YV26 RuBisCo large chain 7505 O98722 RuBisCo large subunit 604 P21239 RuBisCO large subunit binding protein 2403 B1PIE1 RuBisCo oxygenase activase alpha 2 2402 A9UHW7 RuBisCo oxygenase activase small isoform 03 Cell growth/division 7803 C0L3G8 Mother of FT and TFL1 like protein 04 Transcription 1108 K9LNQ8 DNA directed RNA polymerase 6706 D4P3L6 Ndly protein 04.1901 General TFs 2401 Q20BK8 Class III homeodomain leucine zipper protein 6810 A8HQJ4 Global transcription factor 6807 K4CPZ3 Transcription factor KAN2 807 A1IKZ7 Transcription factor Tbx4 Fragment 04.1904 Specific TFs 2404 D7THU3 Agamous-like MADS-box protein AGL15-like 05 Protein synthesis 05.01 Ribosomal proteins 3804 K9LMH5 50S ribosomal protein L14 05.04 Translation factors 8501 H1ZY27 Putative elongation factor 1 alpha 6813 Q1HPA9 Elongation factor 2 6803 O23755 Elongation factor 2 7703 P86349 Elongation factor G

54/5.05 63/5.13 67/5.36 66/5.21 63/5.32 50/5.38 60/5.36 58/5.23 60/5.48 62/5.6

56/4.83 55/4.82 56/4.83 56/4.82 55/4.82 50/4.72 52/4.88 52/4.88 50/4.72 50/4.72

635.21 11218.78 192.64 1317.53 3390.77 286.72 21867.13 19197.38 4631.8 399.81

10 24 9 17 19 3 57 46 25 6

0.34±0.24 0.72±0.37 0.45±0.12 0.38±0.25 0.32±0.17 0.47±0.26 2.53±1.21 0.65±0.15 0.40±0.14 0.07±0.05

18/7.73 22/7.12 23/6.75 30/6.42 39/5.93 48/6.01 110/7.58 30/6.86 17/5.31 29/5.1 16/5.34 29/5.1 26/5.78 35/6.24 23/6.12 35/6.24 41/6.11 16/8.58 39/5.57 16/8.58 55/7.8 53/6.3 41/6.8 53/5.97 70/7.02 49/6.29 50/6.15 53/5.97 83/6.74 49/6.29 250/6.9 20/6.94 102/6.25 49/6.29 55/6.8 53/6.3 55/6.9 53/5.97 57/6.63 53/6.3 58/6.7 53/6.3 58/6.55 53/6.3 180/6.83 46/5.71 66/5.21 53/5.98 57/7.07 22/5.4 54/7.35 20/6.94 57/6.47 25/5.72 58/7.42 25/5.72 250/6.83 25/5.72 55/7.58 53/6.42 67/5.05 58/4.64 42/5.56 16/8.58 45/5.56 48/8.18

366.27 135.46 1256.55 282.38 13220.84 2232.48 1100.02 2461.32 858.56 337.19 307.84 417.05 5734.4 709.25 219.14 1207.35 139.9 6709.48 12104.97 2349.38 5352.14 1590.2 240.85 70.41 12207.37 3631.08 1666.18 1357.03 962.31 253.89 134.75 606.05 458.21

2 2 9 5 8 3 8 11 2 2 10 8 4 11 6 4 4 40 87 28 31 12 6 3 12 6 10 3 6 8 3 2 9

0.29±0.08 0.17±0.05 2.09±1.26 0.26±0.01 0.27±0.04 0.27±0.15 0.82±0.12 0.22±0.05 0.24±0.06 0.19±0.10 0.07±0.08 0.12±0.08 0.10±0.06 0.10±0.05 0.08±0.06 0.05±0.03 0.03±0.02 26.91±2.63 10.49±2.79 7.80±2.98 3.66±3.72 1.02±0.12 0.29±0.24 0.24±0.04 5.45±3.69 0.43±0.21 0.41±0.38 0.27±0.23 0.15±0.06 0.30±0.07 0.34±0.10 0.29±0.06 0.08±0.05

110/7.5 24/10.1

259.96

3

0.22±0.02

13/5.2 90/7.2

78/9.66 47/9.01

77.05 83.36

2 3

0.14±0.10 0.29±0.11

50/5.5 110/7.4 115/7.3 133/5.0

92/5.86 126/4.97 16/6.76 17/9.37

71.69 86.4 137.04 74.11

6 4 2 2

1.93±0.99 0.11±0.01 0.12±0.04 0.14±0.04

48/5.7

21/9.5

185.27

5

0.13±0.09

103/6.1 14/9.39

199.07

5

0.11±0.09

53/7.8 18/6.61 110/7.00 9/9.41 110/7.08 94/5.88 90/7.9 12/5.69

291.32 318.95 200.78 595.6

2 2 11 3

0.20±0.11 0.16±0.05 0.14±0.05 0.04±0.01



333

06 Protein destination and storage 06.01 Folding and stability 1702 M0VTF2 70 kDa heat shock protein 2602 A7Y7L7 GloEL protein chaperonin 60kDa 3705 M1B4C8 Heat shock cognate 70 kDa 1706 P27322 Heat shock cognate 70 kDa protein 2 1705 J3NEA8 Heat shock protein 70 3704 D8RBE2 Heat shock protein 70 2707 A2XF47 Heat shock protein 70 06.13 Proteolysis 3809 P84565 ATP dependent Clp protease 3807 P84565 ATP dependent Clp protease 3810 P84565 ATP dependent Clp protease 3803 P84565 ATP dependent Clp protease 2706 O82150 ATP dependent zinc metalloprotease FTSH 4301 I1LZJ2 CAAX prenyl protease 2 7807 I1MXB2 Glycine dehydrogenase [decarboxylating] 8801 O49954 Glycine dehydrogenase decarboxylating 06.20 Storage proteins 6707 Q8LPD3 Conlinin n 1 2405 Q39772 Ginnacin 2406 Q39772 Ginnacin 1202 Q39772 Ginnacin 6302 Q39772 Ginnacin 1214 Q39772 Ginnacin 6309 Q39772 Ginnacin 3701 Q39772 Ginnacin 2506 Q39772 Ginnacin 2407 Q39772 Ginnacin 2502 Q39772 Ginnacin 7601 Q39772 Ginnacin 3706 Q39772 Ginnacin 2802 Q39772 Ginnacin 2708 Q39772 Ginnacin 2704 B7U6L4 Globulin 3 10 Signal transduction 01 Receptors 2715 G7I4I2 Pheromone receptor like protein 11 Disease/defense 2208 Q6S9Z8 ASR protein 2204 Q6S9Z8 ASR protein 2416 Q6S9Z8 ASR protein 2209 Q6S9Z8 ASR protein 2302 Q6S9Z8 ASR protein 11.05 Stress (and defence) responses 5204 B8YNY1 Ascorbate peroxidase 6104 B8YNY1 Ascorbate peroxidase 3101 B8YNX9 Superoxide dismutase 7105 A6N9I6 Superoxide dismutase 5105 B8YNX9 Superoxide dismutase 11.06 Detoxification 4305 Q01908 ATP synthase gamma chain 1 4309 Q01908 ATP synthase gamma chain 1 4401 A0MQ80 Monodehydroascorbate reductase 1113 G7II43 NAD P H quinone oxidoreductase 3305 F6GY60 Peroxidase 12-like 1107 H6VND7 Peroxiredoxin 2

Page 16 of 25

85/5.01 67/5.5 82/6.2 86/5.44 86/5.4 82/6.2 87/6.0

62/9.45 18/10.53 63/4.85 71/4.88 71/7.9 72/4.98 45/4.93

539.93 1436.29 105.64 1701.94 1145.28 83.65 111.68

9 5 4 13 14 4 5

0.50±0.15 0.26±0.11 0.14±0.05 0.34±0.11 0.32±0.25 0.11±0.03 0.08±0.06

103/6.3 98/6.1 97/6.3 104/6.1 79/6.0 36/6.4 110/7.6 110/7.9

8/4.17 8/4.17 8/4.17 8/4.17 77/5.65 38/8.54 114/7.05 113/6.53

696.96 743.91 1181.43 274.81 370.31 96.2 589.17 124.33

3 3 4 2 12 2 10 7

0.19±0.04 0.19±0.05 0.09±0.06 0.09±0.04 0.16±0.03 0.26±0.03 0.12±0.13 0.08±0.05

75/7.3 42/5.8 46/5.8 25/5.1 32/7.1 18/5.0 37/7.2 73/6.1 50/5.9 47/5.9 49/5.7 57/7.5 74/6.24 103/6.0 77/6.0 81/5.8

19/7.49 51/8.0 51/8.0 51/8.0 51/8.0 51/8.0 51/8.0 51/8.0 51/8.0 51/8.0 51/8.0 51/8.0 51/8.0 51/8.0 51/8.0 66/7.76

3504.32 95.72 65.8 93.6 93.06 134.23 95.07 92.51 70.87 75.19 63.87 50.7 66.55 88.29 88.29 138.55

2 3 7 4 4 5 4 5 6 4 4 3 5 4 4 6

0.03±0.01 0.69±0.18 0.50±0.06 0.40±0.24 0.35±0.05 0.35±0.34 0.24±0.17 0.10±0.09 0.16±0.03 0.15±0.09 0.15±0.04 0.12±0.05 0.16±0.03 0.04±0.03 0.01±0.01 0.08±0.03

72/5.9

34/10.0

52.99

3

0.12±0.02

25/5.6 25/5.5 42/5.9 24/5.6 33/5.5

20/5.2 20/5.2 20/5.2 20/5.2 20/5.2

5891.91 3348.01 92.3 1910.87 97.93

8 2 2 3 2

0.70±0.42 0.61±0.07 0.52±0.52 0.41±0.36 0.11±0.01

18/6.8 17/6.8 14/6.2 14/7.5 14/6.4

27/5.8 27/5.8 27/6.9 26/9.3 27/6.9

3561.47 2035.51 260.77 293.49 1243.84

16 13 4 4 6

0.20±0.03 0.06±0.05 0.28±0.13 0.08±0.09 0.03±0.02

36/6.5 33/6.5 47/6.3 15/5.1 49/6.1 13/5.1

41/8.1 41/8.1 47/5.2 30/9.4 36/5.4 30/7.1

354.9 145.2 248.68 74.35 720.67 1543.6

2 2 5 2 2 6

0.24±0.03 0.22±0.10 0.06±0.03 0.30±0.39 0.14±0.02 0.10±0.08


Page 17 of 25

334


12 Protein of unknown function (PUF) 8010 M5XFL4 Chloro diff and palisade dev-related protein 4103 E4MXM1 mRNA clone RTFL01 39 N23 1201 Q5QNF4 Os01g0217100 protein 3403 Q5QNF4 Os01g0217100 protein 7301 Q5QNF4 Os01g0217100 protein 6804 B9I1Z7 Predicted protein 2803 A9PIA4 Predicted protein n 1501 G7KFS4 Putative uncharacterized protein 5101 M0TUE2 Uncharacterized protein 6702 M4ECD1 Uncharacterized protein 915 M0U8E4 Uncharacterized protein 6701 M4EFH1 Uncharacterized protein 4601 M5WLX4 Uncharacterized protein 2605 K3XJ71 Uncharacterized protein 3408 M1CP73 Zinc finger CCCH domain-containing protein 11 20 Secondary metabolism 20.1 Phenylpropanoids/phenolics 1206 K7YEU4 Caffeoyl CoA O methyltransferase 303 G3EKI9 Cinnamoyl CoA reductase 20.07 Flavonoids 4306 Q5XLY0 Anthocyanidin reductase 6405 Q6RIB2 Chalcone synthase 6204 M1T9X3 Isoflavone reductase like protein 20.99 Others 5401 I1NIQ0 GDP-mannose 3,5-epimerase 2210 F6H9A9 Thiamine thiazole synthase 1 chloroplastic

9/7.7 17/6.6 23/5.1 43/6.0 42/7.6 110/7.2 110/6.0 55/5.1 14/6.7 105/7.0 250/5.0 85/7.0 62/6.4 70/5.8 40/6.2

30/9.4 50/5.7 19/10.2 19/10.2 19/10.2 26/9.0 24/7.5 19/10.2 44/6.4 41/4.9 17/9.4 15/5.0 72/5.2 39/12.1 48/6.8

1213.14 154.17 422.25 607.16 693.39 76.7 445.74 147.34 84.73 104.43 183.62 347.02 44.04 150.54 131.11

2 2 2 2 4 3 2 2 2 2 2 2 2 3 4

0.05±0.05 0.13±0.11 0.35±0.41 0.27±0.02 0.06±0.05 0.06±0.04 0.11±0.04 0.31±0.24 0.19±0.09 0.15±0.05 0.15±0.06 0.11±0.01 0.09±0.04 0.07±0.01 0.21±0.21

21/5.4 36/5.1

29/5.5 36/7.1

537.88 57.26

4 2

0.19±0.03 0.51±0.15

36/6.6 41/7.3 28/7.4

37/5.5 43/6.3 33/6.2

1405.2 174.41 445.65

8 6 5

0.12±0.03 0.28±0.14 0.20±0.16

49/6.7 27/5.7

31/5.6 37/5.7

1077.03 690.37

10 6

0.12±0.05 0.19±0.01

335



336

References

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Establishing a Leaf Proteome Reference Map for Ginkgo biloba

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