THE PROTEOME OF OLIVE SEEDS AND THE INVISIBLE PROTEOME OF OLIVE OILS AS DETECTED

VIA COMBINATIONAL PEPTIDE LIGAND LIBRARY CAPTUREClara Estevea, Alfonsina D’Amatob, M. Luisa Marinaa, M. Concepción Garcíaa , Pier Giorgio Righettib

aDepartment of Analytical Chemistry, University of Alcalá. Alcalá de Henares (Madrid), Spainb Department of Chemistry, Materials and Chemical Engineering “Giulio Natta”, Politecnico di Milano. Milan, Italy

♠ Olive fruit and olive oil proteins have been scarcely investigated, despite their highly informative value and role in food stability and allergenicity.♠ There is a high difficulty in extracting proteins from vegetable matrices rich in lipids. The use of combinational peptide ligand libraries (CPLLs) at pH 7.4 and 2.2 has been employed in the extraction protocols.

The main objective of this work is to establish a suitable method for the isolation of proteins from olive seed and olive oil based on the application of the most

improved CPLL technology and for the detection of olive fruit and olive oil proteome.

Protein extraction

Experimental conditions

♠ Chromatographic conditions- Column: Acclaim PepMap 100 C18 (20 mm x 100 µm)

- Mobile phases: A (0.1% (v/v) FA, 2% (v/v) AcN in water)

B (0.1% (v/v) FA, 2% (v/v) water in AcN)- Gradient: 4% to 40 % phase A in 30 min.- Flow rate: 300 µL/min- Injected volume: 8 µL♠ ESI-LIT

- Equipment: LTQ XL (Thermo Scientific)

MS/MS Analysis

SoftwareMASCOT Search Engine (Version 2.3.01)Database: Uniprot_viridiplantae (30264 residues, 184678199 sequences)

Precipitate redisolution in 4% SDS 25 mM DTT, 100

ºC

+400 mL 25% TCA/Acetone

Precipitation MeOH/CH3Cl

Olive oil(400 mL)

SDS-PAGE-Silver staining

M. C. García thanks financial support from project CTQ2009-11252 (Ministry of Science and Innovation, Spain). M. L. Marina and M. C. García thank financial support from project S-2009/AGR-1464 (Comunidad Autónoma of Madrid, Spain). C. Esteve thanks the University of Alcalá for her pre-doctoral and mobility grants and additionally FEBS (Federation of European Biochemical Societies) for a short-term fellowship for a stage at the Politecnico di Milano.

♠ It has been possible to identify 61 unique gene products in the olive seed, representing an important increment in the exploration of seed species. ♠ A high number of isoforms of histones and globulin seed storage proteins, and an oleosin of 17,2 kDa. ♠ It has been confirmed that CPLL treatment permits identifying a much larger number of species than in control, untreated sample, even in such recalcitrant tissues as plant material. ♠ For olive oil, we could assess the presence of very fine bands in SDS-PAGE gels, centered in the 10-30 kDa regions, whose identification is now in progress. Such species are found only in trace amounts, though, and can only be visualized via silver staining, thus their identification might not represent an easy task.

Olive seed

SDS-PAGE-Coomassie blue

Tris-HCl (pH 7.4), NaCl, EDTA, DTT, SDS, protease

inhibitor cocktail

CPLLs(pH 7.4)

Urea, thiourea, CHAPS, protease inhibitor cocktail

Tris-HCl (pH 7.4), NaCl, CHAPS, protease inhibitor

cocktail

CPLLs(pH 7.4)

CPLLs(pH 2.2)

Proteins SDS-PAGE separation, digestion, and analysis

Controls Captures

250150

KDa

10075

50

37

2520

1510

Mr

250150

KDa

100755037

2520

15

10

MrOlive oil proteins

Trypsin digestion

nLC-MS/MS(LTQ-XL)

Protein identification

31 11 19

Total proteins: 61

CPLLcapture

CONTROLS

INTRODUCTION OBJECTIVE

EXPERIMENTAL

RESULTS

CONCLUSIONS

ACKNOWLEDMENTS

Accession numbers Protein name Mascot score Mr N peptide CTRL CPLLstr|B9H8M5|B9H8M5_POPTR Predicted protein OS=Populus trichocarpa GN=POPTRDRAFT_818720 PE=3 SV=1 747 54432 1 x

tr|B9GS11|B9GS11_POPTR Predicted protein OS=Populus trichocarpa GN=POPTRDRAFT_830076 PE=3 SV=1 496 54751 1 xtr|C0L8H1|C0L8H1_ORYSJ GluB-5 long variant OS=Oryza sativa subsp. japonica PE=2 SV=1 331 57153 1 xtr|B6UH77|B6UH77_MAIZE Histone H3 OS=Zea mays PE=2 SV=1 186 15344 1 xtr|D0F1P7|D0F1P7_9ROSI Aspartic proteinase 1 OS=Castanea mollissima PE=2 SV=1 182 56802 2 xtr|Q8LGL8|Q8LGL8_OLEEU Oleosin OS=Olea europaea subsp. europaea PE=2 SV=1 167 17184 3 xtr|Q2XSW6|Q2XSW6_SESIN 11S globulin isoform 4 OS=Sesamum indicum PE=2 SV=1 153 52993 2 xsp|P85925|UP18_PSEMZ Unknown protein 18 (Fragment) OS=Pseudotsuga menziesii PE=1 SV=1 146 1393 1 xtr|C0PQH4|C0PQH4_PICSI Histone H3 OS=Picea sitchensis PE=2 SV=1 136 15302 1 xtr|B6T0G9|B6T0G9_MAIZE Histone H4 OS=Zea mays PE=3 SV=1 126 12087 4 xtr|D8RNW6|D8RNW6_SELML Histone H2B (Fragment) OS=Selaginella moellendorffii GN=SELMODRAFT_18569 PE=3 SV=1 122 11397 2 xtr|C0P3K6|C0P3K6_MAIZE Putative uncharacterized protein OS=Zea mays PE=2 SV=1 119 55203 1 xsp|P19084|11S3_HELAN 11S globulin seed storage protein G3 OS=Helianthus annuus GN=HAG3 PE=3 SV=1 118 55995 1 xtr|B6UGJ0|B6UGJ0_MAIZE Globulin-1 S allele OS=Zea mays PE=2 SV=1 113 50275 2 xtr|D8SSH2|D8SSH2_SELML Histone H2B OS=Selaginella moellendorffii GN=SELMODRAFT_159683 PE=3 SV=1 99 15637 2 xtr|B9GVX4|B9GVX4_POPTR Histone H3 (Fragment) OS=Populus trichocarpa GN=POPTRDRAFT_415475 PE=3 SV=1 95 9467 6 xtr|F4YF90|F4YF90_CAMSI Histone H2B OS=Camellia sinensis PE=2 SV=1 95 15328 3 xsp|P06353|H33_HORVU Histone H3.3 (Fragment) OS=Hordeum vulgare PE=3 SV=1 94 9284 6 xtr|D7SZN0|D7SZN0_VITVI Putative uncharacterized protein OS=Vitis vinifera GN=VIT_05s0049g01780 PE=4 SV=1 94 26580 1 xtr|Q2Q4Y5|Q2Q4Y5_MEDTR Em6 OS=Medicago truncatula PE=4 SV=1 94 10734 1 xtr|A5C4Y7|A5C4Y7_VITVI Putative uncharacterized protein OS=Vitis vinifera GN=VITISV_009829 PE=4 SV=1 92 43342 1 xtr|B9ICY2|B9ICY2_POPTR Predicted protein OS=Populus trichocarpa GN=POPTRDRAFT_734053 PE=4 SV=1 92 21772 1 xtr|Q4JKA5|Q4JKA5_RHEAU Histone H3 OS=Rheum australe PE=2 SV=1 91 15339 5 xtr|Q6EUQ1|Q6EUQ1_ORYSJ Os02g0176100 protein OS=Oryza sativa subsp. japonica GN=OJ1077_E05.22-1 PE=2 SV=1 88 86338 1 xtr|A5AGR6|A5AGR6_VITVI Putative uncharacterized protein OS=Vitis vinifera GN=VITISV_032539 PE=4 SV=1 84 67973 1 xtr|A9NX28|A9NX28_PICSI Putative uncharacterized protein OS=Picea sitchensis PE=2 SV=1 83 64563 1 xtr|Q7XH13|Q7XH13_ORYSJ Transposon protein, putative, CACTA, En/Spm sub-class OS=Oryza sativa subsp. japonica GN=LOC_Os10g04760 PE=4 SV=1 73 9600 1 xtr|B4G227|B4G227_MAIZE Putative uncharacterized protein OS=Zea mays PE=2 SV=1 72 67783 1 xtr|B9GNF8|B9GNF8_POPTR Predicted protein OS=Populus trichocarpa GN=POPTRDRAFT_830563 PE=4 SV=1 70 41910 1 xtr|Q40347|Q40347_MAGSL Globulin OS=Magnolia salicifolia PE=2 SV=1 70 53858 1 xtr|B9GKQ2|B9GKQ2_POPTR Predicted protein OS=Populus trichocarpa GN=POPTRDRAFT_548528 PE=3 SV=1 69 63193 1 xtr|Q5DWG7|Q5DWG7_CONCI Histone H3 OS=Conocephalum conicum GN=H3 gene PE=3 SV=1 68 15419 1 xtr|D8QUA3|D8QUA3_SELML Histone H3 OS=Selaginella moellendorffii GN=SELMODRAFT_103546 PE=3 SV=1 67 15392 5 xtr|Q0DIG5|Q0DIG5_ORYSJ Os05g0391500 protein OS=Oryza sativa subsp. japonica GN=Os05g0391500 PE=4 SV=1 64 111163 1 xtr|F1LKN0|F1LKN0_PHAVU Aspartic acid protease OS=Phaseolus vulgaris PE=2 SV=1 63 56341 2 xtr|F4YF87|F4YF87_CAMSI Histone H4 OS=Camellia sinensis PE=2 SV=1 62 14989 1 xsp|P0CG89|H4_SOYBN Histone H4 OS=Glycine max PE=3 SV=1 61 11402 1 xtr|B9ILZ3|B9ILZ3_POPTR Histone H4 OS=Populus trichocarpa GN=HFO906 PE=3 SV=1 61 10203 1 xtr|Q8H786|Q8H786_ARATH Putative uncharacterized protein (Fragment) OS=Arabidopsis thaliana PE=2 SV=1 60 12468 1 xtr|F2EJZ5|F2EJZ5_HORVD Predicted protein OS=Hordeum vulgare var. distichum PE=2 SV=1 59 21637 1 xtr|E9MZ24|E9MZ24_9CHLO Histone H3 (Fragment) OS=Cladophora albida PE=2 SV=1 58 13808 5 xsp|P85487|UP03_PINHA Unknown protein 3 (Fragment) OS=Pinus halepensis PE=1 SV=1 57 1158 1 xtr|B3H460|B3H460_ARATH Uncharacterized protein OS=Arabidopsis thaliana GN=At3g49645 PE=4 SV=1 57 25171 1 xtr|C1E478|C1E478_MICSR Predicted protein OS=Micromonas sp. (strain RCC299 / NOUM17) GN=MICPUN_105456 PE=4 SV=1 57 65777 2 xsp|Q9XHP0|11S2_SESIN 11S globulin seed storage protein 2 OS=Sesamum indicum PE=2 SV=1 56 52083 1 xtr|A5BD19|A5BD19_VITVI Putative uncharacterized protein OS=Vitis vinifera GN=VITISV_024449 PE=4 SV=1 55 82103 1 xtr|Q9LRB1|Q9LRB1_SCODU Copalyl diphosphate OS=Scoparia dulcis GN=cps PE=2 SV=1 55 94545 1 xtr|B2CGM5|B2CGM5_WHEAT Triticin OS=Triticum aestivum PE=2 SV=1 54 65241 1 xtr|D8TLR3|D8TLR3_VOLCA Putative uncharacterized protein OS=Volvox carteri f. nagariensis GN=VOLCADRAFT_87600 PE=4 SV=1 54 109716 1 xtr|B6RF08|B6RF08_SACOF Brassinosteroid insensitive1-associated receptor kinase 1 OS=Saccharum officinarum GN=BAK1 PE=2 SV=1 53 69894 1 xtr|B9I6P2|B9I6P2_POPTR Chromatin remodeling complex subunit OS=Populus trichocarpa GN=CHR945-1 PE=4 SV=1 53 257289 1 xtr|D7M9P0|D7M9P0_ARALL Putative uncharacterized protein OS=Arabidopsis lyrata subsp. lyrata GN=ARALYDRAFT_353547 PE=4 SV=1 53 64630 1 xtr|C0JDD5|C0JDD5_9BRAS At4g14370-like protein (Fragment) OS=Capsella grandiflora PE=4 SV=1 52 22013 1 xtr|E0WCL8|E0WCL8_CARTI Putative uncharacterized protein OS=Carthamus tinctorius PE=2 SV=1 52 15789 1 xtr|Q013L7|Q013L7_OSTTA Glucose-repressible alcohol dehydrogenase transcriptional effector CCR4 and related proteins (ISS) OS=Ostreococcus tauri GN=Ot08g01740 PE=4 SV=1 51 95987 1 xtr|B9H8B1|B9H8B1_POPTR Predicted protein OS=Populus trichocarpa GN=POPTRDRAFT_715001 PE=4 SV=1 51 48957 1 xtr|C1EBG0|C1EBG0_MICSR Predicted protein OS=Micromonas sp. (strain RCC299 / NOUM17) GN=MICPUN_60417 PE=4 SV=1 51 28360 1 xtr|B9FES4|B9FES4_ORYSJ Putative uncharacterized protein OS=Oryza sativa subsp. japonica GN=OsJ_14499 PE=4 SV=1 50 57062 1 xtr|D8RTN9|D8RTN9_SELML Putative uncharacterized protein (Fragment) OS=Selaginella moellendorffii GN=SELMODRAFT_30949 PE=4 SV=1 50 60116 1 xtr|Q9AU64|Q9AU64_ELAGV 7S globulin OS=Elaeis guineensis var. tenera GN=GLO7A PE=2 SV=1 48 67008 1 xtr|D8RZB7|D8RZB7_SELML Putative uncharacterized protein OS=Selaginella moellendorffii GN=SELMODRAFT_104848 PE=4 SV=1 47 41486 1 x

Olive seed Olive oil

Coomassie Blue staining Silver staining

Venn Diagram Gene Ontology

The various captures with CPLLs permitted the additional

identification of 31 unique gene products

It demonstrates the role of seed in containing genetic material for plant development.

OLIVE SEED

OLIVE OIL

FUTURE ANALYSIS IN A MORE SENSITIVE

EQUIPMENT