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THE PROTEOME OF OLIVE SEEDS AND THE INVISIBLE PROTEOME OF OLIVE OILS AS DETECTED VIA COMBINATIONAL PEPTIDE LIGAND LIBRARY CAPTURE Clara Esteve a , Alfonsina D’Amato b , M. Luisa Marina a , M. Concepción García a , Pier Giorgio Righetti b a Department of Analytical Chemistry, University of Alcalá. Alcalá de Henares (Madrid), Spain b 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 C 18 (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 Software MASCOT 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/CH 3 Cl 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 C ontrols C aptures 250 150 KDa 100 75 50 37 25 20 15 10 Mr 250 150 KDa 100 75 50 37 25 20 15 10 Mr O live oil proteins Trypsin digestion nLC-MS/MS (LTQ-XL) Protein identification 31 11 19 T otalproteins:61 CPLL captu re CONTROLS INTRODUCTION OBJECTIVE EXPERIMENTAL RESULTS CONCLUSIONS ACKNOWLEDMENTS A ccession num bers Protein nam e M ascotscore M r N peptide CTRL CPLLs tr|B9H8M 5|B9H8M 5_PO PTR Predicted protein O S=Populus trichocarpa GN =PO PTRD RA FT_818720 PE=3 SV=1 747 54432 1 x tr|B9GS11|B9GS11_PO PTR Predicted protein O S=Populus trichocarpa GN =PO PTRD RA FT_830076 PE=3 SV=1 496 54751 1 x tr|C0L8H 1|C0L8H 1_O RYSJ GluB-5 long variantO S=O ryza sativa subsp. japonica PE=2 SV=1 331 57153 1 x tr|B6UH77|B6U H77_M A IZE H istone H 3 O S=Zea m ays PE=2 SV=1 186 15344 1 x tr|D 0F1P7|D 0F1P7_9ROSI A spartic proteinase 1 OS=Castanea mollissima PE=2 SV=1 182 56802 2 x tr|Q 8LGL8|Q 8LGL8_O LEEU O leosin O S=O lea europaea subsp. europaea PE=2 SV=1 167 17184 3 x tr|Q2XSW 6|Q2XSW 6_SESIN 11S globulin isoform 4 O S=Sesam um indicum PE=2 SV=1 153 52993 2 x sp|P85925|U P18_PSEM Z U nknow n protein 18 (Fragm ent)O S=Pseudotsuga m enziesii PE=1 SV=1 146 1393 1 x tr|C0PQ H 4|C0PQ H 4_PICSI H istone H 3 O S=Picea sitchensis PE=2 SV=1 136 15302 1 x tr|B6T0G9|B6T0G9_M A IZE H istone H 4 O S=Zea m ays PE=3 SV=1 126 12087 4 x tr|D8RNW 6|D 8RN W 6_SELM L Histone H2B (Fragment)O S=Selaginella moellendorffii GN=SELM ODRA FT_18569 PE=3 SV=1 122 11397 2 x tr|C0P3K6|C0P3K6_M A IZE Putative uncharacterized protein O S=Zea m ays PE=2 SV=1 119 55203 1 x sp|P19084|11S3_HELA N 11S globulin seed storage protein G3 O S=H elianthus annuus GN =H A G3 PE=3 SV=1 118 55995 1 x tr|B6U GJ0|B6U GJ0_M A IZE Globulin-1 S allele O S=Zea m ays PE=2 SV=1 113 50275 2 x tr|D 8SSH 2|D 8SSH 2_SELM L H istone H 2B O S=Selaginella m oellendorffii GN =SELM ODRA FT_159683 PE=3 SV=1 99 15637 2 x tr|B9GVX 4|B9GVX 4_PO PTR H istone H 3 (Fragm ent)O S=Populus trichocarpa GN =PO PTRD RA FT_415475 PE=3 SV=1 95 9467 6 x tr|F4YF90|F4YF90_CA M SI H istone H 2B O S=Cam ellia sinensis PE=2 SV=1 95 15328 3 x sp|P06353|H 33_H O RVU H istone H 3.3 (Fragm ent)O S=H ordeum vulgare PE=3 SV=1 94 9284 6 x tr|D 7SZN 0|D 7SZN 0_VITVI Putative uncharacterized protein O S=Vitis vinifera GN =VIT_05s0049g01780 PE=4 SV=1 94 26580 1 x tr|Q2Q4Y5|Q2Q4Y5_M EDTR Em 6 O S=M edicago truncatula PE=4 SV=1 94 10734 1 x tr|A 5C4Y7|A 5C4Y7_VITVI Putative uncharacterized protein O S=Vitis vinifera GN =VITISV_009829 PE=4 SV=1 92 43342 1 x tr|B9ICY2|B9ICY2_PO PTR Predicted protein O S=Populus trichocarpa GN =PO PTRD RA FT_734053 PE=4 SV=1 92 21772 1 x tr|Q 4JKA 5|Q4JKA 5_RH EA U H istone H 3 O S=Rheum australe PE=2 SV=1 91 15339 5 x tr|Q 6EU Q 1|Q 6EU Q 1_O RYSJ O s02g0176100 protein O S=O ryza sativa subsp. japonica GN =O J1077_E05.22-1 PE=2 SV=1 88 86338 1 x tr|A 5A GR6|A 5A GR6_VITVI Putative uncharacterized protein O S=Vitis vinifera GN =VITISV_032539 PE=4 SV=1 84 67973 1 x tr|A 9N X28|A 9N X 28_PICSI Putative uncharacterized protein O S=Picea sitchensis PE=2 SV=1 83 64563 1 x tr|Q 7X H 13|Q 7X H 13_O RYSJ Transposon protein, putative, CA CTA , En/Spm sub-class O S=O ryza sativa subsp. japonica GN =LO C_O s10g04760 PE=4 SV=1 73 9600 1 x tr|B4G227|B4G227_M A IZE Putative uncharacterized protein O S=Zea m ays PE=2 SV=1 72 67783 1 x tr|B9GN F8|B9GN F8_PO PTR Predicted protein O S=Populus trichocarpa GN =PO PTRD RA FT_830563 PE=4 SV=1 70 41910 1 x tr|Q40347|Q40347_M A GSL Globulin O S=M agnolia salicifolia PE=2 SV=1 70 53858 1 x tr|B9GK Q 2|B9GK Q 2_PO PTR Predicted protein O S=Populus trichocarpa GN =PO PTRD RA FT_548528 PE=3 SV=1 69 63193 1 x tr|Q5DW G7|Q 5D W G7_CONCI H istone H 3 O S=Conocephalum conicum GN =H 3 gene PE=3 SV=1 68 15419 1 x tr|D8QUA 3|D8QUA 3_SELM L H istone H 3 O S=Selaginella moellendorffii GN =SELM ODRA FT_103546 PE=3 SV=1 67 15392 5 x tr|Q 0D IG5|Q 0D IG5_O RYSJ O s05g0391500 protein O S=O ryza sativa subsp. japonica GN =O s05g0391500 PE=4 SV=1 64 111163 1 x tr|F1LKN 0|F1LK N 0_PH A VU A spartic acid protease O S=Phaseolus vulgaris PE=2 SV=1 63 56341 2 x tr|F4YF87|F4YF87_CA M SI H istone H 4 O S=Cam ellia sinensis PE=2 SV=1 62 14989 1 x sp|P0CG89|H 4_SO YBN H istone H 4 O S=Glycine m axPE=3 SV=1 61 11402 1 x tr|B9ILZ3|B9ILZ3_PO PTR H istone H 4 O S=Populus trichocarpa GN =H FO 906 PE=3 SV=1 61 10203 1 x tr|Q8H786|Q8H786_A RA TH Putative uncharacterized protein (Fragm ent)O S=A rabidopsis thaliana PE=2 SV=1 60 12468 1 x tr|F2EJZ5|F2EJZ5_H O RVD Predicted protein O S=H ordeum vulgare var. distichum PE=2 SV=1 59 21637 1 x tr|E9M Z24|E9M Z24_9CH LO H istone H 3 (Fragm ent)O S=Cladophora albida PE=2 SV=1 58 13808 5 x sp|P85487|UP03_PINHA U nknow n protein 3 (Fragm ent)O S=Pinus halepensis PE=1 SV=1 57 1158 1 x tr|B3H460|B3H460_A RA TH U ncharacterized protein O S=A rabidopsisthaliana GN=A t3g49645 PE=4 SV=1 57 25171 1 x tr|C1E478|C1E478_M ICSR Predicted protein O S=M icrom onas sp. (strain RCC299 /N O U M 17)GN =M ICPU N _105456 PE=4 SV=1 57 65777 2 x sp|Q 9XH P0|11S2_SESIN 11S globulin seed storage protein 2 O S=Sesam um indicum PE=2 SV=1 56 52083 1 x tr|A 5BD19|A 5BD 19_VITVI Putative uncharacterized protein O S=Vitis vinifera GN =VITISV_024449 PE=4 SV=1 55 82103 1 x tr|Q 9LRB1|Q 9LRB1_SCO D U Copalyl diphosphate O S=Scoparia dulcis GN =cps PE=2 SV=1 55 94545 1 x tr|B2CGM 5|B2CGM 5_W H EA T Triticin O S=Triticum aestivum PE=2 SV=1 54 65241 1 x tr|D 8TLR3|D 8TLR3_VO LCA Putative uncharacterized protein O S=Volvoxcarteri f. nagariensis GN =VO LCA DRA FT_87600 PE=4 SV=1 54 109716 1 x tr|B6RF08|B6RF08_SA COF Brassinosteroid insensitive1-associated receptorkinase 1 OS=Saccharum officinarum GN=BA K 1 PE=2 SV=1 53 69894 1 x tr|B9I6P2|B9I6P2_POPTR Chrom atin rem odeling com plexsubunitO S=Populus trichocarpa GN =CH R945-1 PE=4 SV=1 53 257289 1 x tr|D 7M 9P0|D7M 9P0_A RA LL Putative uncharacterized protein O S=A rabidopsis lyrata subsp. lyrata GN =A RA LYDRA FT_353547 PE=4 SV=1 53 64630 1 x tr|C0JD D 5|C0JD D 5_9BRA S A t4g14370-like protein (Fragm ent)O S=Capsella grandiflora PE=4 SV=1 52 22013 1 x tr|E0W CL8|E0W CL8_CA RTI Putative uncharacterized protein O S=Cartham us tinctorius PE=2 SV=1 52 15789 1 x tr|Q 013L7|Q013L7_O STTA Glucose-repressible alcohol dehydrogenase transcriptional effectorCCR4 and related proteins(ISS)O S=O streococcus tauri GN =O t08g01740 PE=4 SV=1 51 95987 1 x tr|B9H 8B1|B9H 8B1_PO PTR Predicted protein O S=Populus trichocarpa GN =PO PTRD RA FT_715001 PE=4 SV=1 51 48957 1 x tr|C1EBG0|C1EBG0_M ICSR Predicted protein O S=M icrom onas sp. (strain RCC299 /N O U M 17)GN =M ICPU N _60417 PE=4 SV=1 51 28360 1 x tr|B9FES4|B9FES4_O RYSJ Putative uncharacterized protein O S=O ryza sativa subsp. japonica GN =O sJ_14499 PE=4 SV=1 50 57062 1 x tr|D 8RTN 9|D 8RTN 9_SELM L Putative uncharacterized protein (Fragm ent)O S=Selaginella m oellendorffii GN =SELM ODRA FT_30949 PE=4 SV=1 50 60116 1 x tr|Q 9A U64|Q9A U 64_ELA GV 7S globulin O S=Elaeis guineensis var. tenera GN=GLO 7A PE=2 SV=1 48 67008 1 x tr|D 8RZB7|D 8RZB7_SELM L Putative uncharacterized protein O S=Selaginella m oellendorffii GN =SELM ODRA FT_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
