Research article Received: 13 February 2014
Revised: 30 April 2014
Accepted: 19 June 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3422
Study of isobaric grape seed proanthocyanidins by MALDI-TOF MS† Fabiola De Marchi,a Roberta Seraglia,b Laura Molin,b Pietro Traldi,b Antonio Dalla Vedova,a Massimo Gardiman,a Mirko De Rossoa and Riccardo Flaminia* Positive matrix-assisted laser desorption ionization (MALDI) spectra of grape seed raw extracts show the signals of putative proanthocyanidin (PA) oligomers, including those of several sodium and potassium adducts with the same nominal molecular weight. As a result, the MALDI time-of-flight profiles are characterized by several isobaric signals from different PAs. The presence of isobaric PA adducts was studied by sodium and potassium exchange experiments on the seed extracts of six grape varieties. This approach was effective in differentiating PAs with isobaric signals, and 15 different PAs were identified in the MALDI spectra of all samples. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: proanthocyanidins; isobaric; grape seeds, MALDI-TOF; MS
Introduction
826
Study of grape seed proanthocyanidins (PAs) is of great interest because these compounds are not only responsible for some organoleptic properties of wine, particularly bitterness and astringency, but also have free radical scavenging and antioxidizing activity.[1,2] Grape seed tannins are used as additives to improve the organoleptic characteristics of wines,[3] and seed extracts are added to dietary supplements and food preservatives and in the nutraceutical industry.[4] In general, PAs are flavan-3-ol oligomers and polymers with structure composed of (+)-catechin and ( )-epicatechin units linked at C4-C8 and/or C4-C6 positions by interflavanic bonds (B-type compounds) and C2-C7 and C2-C5 ether bonds (A-type; Fig. 1).[5–7] Grape seed PAs are formed of (+)-catechin, ( )-epicatechin and ( )-epicatechin-3-O-gallate units and contain B-type and A-type bonds.[6,8,9] Matrix-assisted laser desorption ionization time-of-flight MS (MALDI-TOF) has been successfully applied to the study of tannins.[10,11] In grape seeds, PAs with up to 7 degrees of polymerization (DP) were identified by Fourier transform-ion cyclotron resonance (FTICR) MS[12,13] and up to 11 by MALDI-TOF.[14] Thiolysis of extracts followed by gel permeation chromatography identified PAs with DPs of up to 18[8,15]; in direct-infusion electrospray ionization spectra, [M 3H]3 signals of PAs up to 28 units were observed.[16] In general, MS does not clearly reveal high molecular weight (MW) compounds, mainly because of ion suppression caused by the high abundance of lower MW species.[11,17–19] The positive MALDI spectra of grape seed raw extracts show the signals of many putative PA oligomers, including those of several isobaric sodium and potassium adducts with the same nominal MW. Consequently, the MALDI-TOF profiles of samples show several signals that can be potentially assigned to different PAs. In this study, sodium and potassium exchange experiments on grape seed extracts were carried out to study the presence of
J. Mass Spectrom. 2014, 49, 826–830
isobaric signals corresponding to different PAs in the MALDI mass spectra. Extracts of six grape varieties were examined.
Materials and methods Samples and sample preparation Seed extracts of six grape varieties were studied: Aramon (Vitis vinifera × unknown variety), Bacò 1 (V. vinifera × V. riparia), Bertille Seyve 4825 (Bertille Seyve 2667 × Seibel 6905), Galibert 238-35 (Seyve Villard 12-401 × V. vinifera), Seibel 8357 (Seibel 6150 × Seibel 5455) and Seyve Villard 12-390 (Seibel 6468 × Seibel 6905). Grape samples were collected in 2010 at full technological ripeness (maximum sugar content) from the five plants of each variety present in the CRA – Viticulture Research Centre Grapevine Germplasm Collection (Susegana, Treviso, Italy) and immediately frozen. Seeds of 20 berries were manually separated from skins and pulps, dried on a paper sheet, weighted and ground with liquid nitrogen by using a mortar. The sample was defatted with hexane (ratio 1:10 w/v) by keeping the sample under stirring at room temperature overnight in the dark. The powder was separated from the solvent by a paper filter and extracted with a methanol/water solution (70 : 30 v/v) in ratio 1 : 10 (w/v).[20] A volume of 1 ml of the extract was reduced to 0.5 ml under vacuum, added with 4.5 ml water then passed through a 360-mg
* Correspondence to: Riccardo Flamini, Consiglio per la Ricerca e la Sperimentazione in Agricoltura – Centro di Ricerca per la Viticoltura (CRA-VIT), Viale XXVIII Aprile 26, 31015 Conegliano, Treviso, Italy. E-mail: riccardo.fl[email protected] †
This article is part of the Journal of Mass Spectrometry special issue entitled “3rd MS Food Day” edited by Gianluca Giorgi.
a CRA-VIT, Viale XXVIII aprile 26, 31015 Conegliano, Treviso, Italy b CNR-IENI, Corso Stati Uniti 4, 35127 Padova, Italy
Copyright © 2014 John Wiley & Sons, Ltd.
Isobaric grape proanthocyanidins by MALDI-TOF MS (70 : 30 v/v). The raw and purified extract solutions were diluted ten times with a methanol/water solution (70 : 30 v/v). In this study, 5 μl of these solutions were mixed with the same volume of matrix solution, and 1 μl of the resulting mixture was deposited directly on the stainless steel sample holder and allowed to dry before introduction into the mass spectrometer. External mass calibration (peptide calibration standard) was based on monoisotopic values of [M + H]+ of bradykinin, angiotensin II, angiotensin I, substance P, bombesin, ACTH clip (1–17), ACTH clip (18–39) and somatostatin 28 at m/z 757.39916, 1046.5420, 1296.6853, 1347.7361, 1619.8230, 2093.0868, 2465.1990 and 3147.4714, respectively. To perform cation exchanging experiments, water solutions of LiCl, NaCl and KCl at a 100-mM concentration were prepared. In this study, 2 μl of the cation salt solution was added to 5 μl of
Figure 1. Structures of grape seed flavanols and proanthocyanidins.
C18 Sep-Pak cartridge (Waters) previously activated by passage of 3 ml methanol and 5 ml water for removing salts and more polar compounds. After sample passage, the cartridge was washed with 3 ml of water, and the purified polyphenolic fraction was eluted with 3 ml of methanol.
Table 1. Peaks of putative isobaric pairs of PAs in the m/z 900–2000 range m/z
Compound
905
Dimer 2G Trimer Trimer 2G Tetramer Trimer 3G Tetramer 1G Tetramer 2G Pentamer Tetramer 3G Pentamer 1G Tetramer 4G Pentamer 2G Pentamer 2G Hexamer Pentamer 3G Hexamer 1G
1193 1345
MALDI-TOF MS
1481
Mass spectrometric measurements were performed using a MALDITOF-TOF UltrafleXtreme (Bruker Daltonics, Bremen, Germany) instrument, equipped with 1-kHz smartbeam II laser (λ = 355 nm) and operating in reflectron positive ion mode. The instrumental conditions employed to analyze molecular species in the m/z range 450–3500 were ion source 1: 25.00 kV; ion source 2: 22.30 kV; lens: 7.70 kV; pulsed ion extraction: 80 ns, reflector: 26.45 kV and reflector 2: 13.45 kV. The matrix solution was prepared by dissolving 10 mg of crystalline 2, 5-dihydroxybenzoic acid in 1 ml of methanol/water solution
1633 1785 1769 1921
Cation +
[M + Na] + [M + K] + [M + Na] + [M + K] + [M + Na] + [M + K] + [M + Na] + [M + K] + [M + Na] + [M + K] + [M + Na] + [M + K] + [M + Na] + [M + K] + [M + Na] + [M + K]
827
Figure 2. MALDI mass spectrum of Bacò 1 grape seeds raw extract.
J. Mass Spectrom. 2014, 49, 826–830
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jms
F. De Marchi et al. the solution containing the purified extract and stirred for about 1 min. After stirring, 5 μL of 2,5-dihydroxybenzoic acid solution was added, and 1 μl of the resulting mixture was deposited directly on the stainless steel sample holder and allowed to dry before introduction into the mass spectrometer.
Results and discussion The positive MALDI-TOF ‘fingerprints’ of grape seed extracts show decreasing peak intensities with increasing m/z values, corresponding to PAs [M + Na]+ and [M + K]+ adducts according to published
data (Fig. 2).[14,21] The signals of PA sodium and potassium adducts with the same nominal MW were identified among the peaks. In general, the more intense peaks observed at m/z
Revised: 30 April 2014
Accepted: 19 June 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3422
Study of isobaric grape seed proanthocyanidins by MALDI-TOF MS† Fabiola De Marchi,a Roberta Seraglia,b Laura Molin,b Pietro Traldi,b Antonio Dalla Vedova,a Massimo Gardiman,a Mirko De Rossoa and Riccardo Flaminia* Positive matrix-assisted laser desorption ionization (MALDI) spectra of grape seed raw extracts show the signals of putative proanthocyanidin (PA) oligomers, including those of several sodium and potassium adducts with the same nominal molecular weight. As a result, the MALDI time-of-flight profiles are characterized by several isobaric signals from different PAs. The presence of isobaric PA adducts was studied by sodium and potassium exchange experiments on the seed extracts of six grape varieties. This approach was effective in differentiating PAs with isobaric signals, and 15 different PAs were identified in the MALDI spectra of all samples. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: proanthocyanidins; isobaric; grape seeds, MALDI-TOF; MS
Introduction
826
Study of grape seed proanthocyanidins (PAs) is of great interest because these compounds are not only responsible for some organoleptic properties of wine, particularly bitterness and astringency, but also have free radical scavenging and antioxidizing activity.[1,2] Grape seed tannins are used as additives to improve the organoleptic characteristics of wines,[3] and seed extracts are added to dietary supplements and food preservatives and in the nutraceutical industry.[4] In general, PAs are flavan-3-ol oligomers and polymers with structure composed of (+)-catechin and ( )-epicatechin units linked at C4-C8 and/or C4-C6 positions by interflavanic bonds (B-type compounds) and C2-C7 and C2-C5 ether bonds (A-type; Fig. 1).[5–7] Grape seed PAs are formed of (+)-catechin, ( )-epicatechin and ( )-epicatechin-3-O-gallate units and contain B-type and A-type bonds.[6,8,9] Matrix-assisted laser desorption ionization time-of-flight MS (MALDI-TOF) has been successfully applied to the study of tannins.[10,11] In grape seeds, PAs with up to 7 degrees of polymerization (DP) were identified by Fourier transform-ion cyclotron resonance (FTICR) MS[12,13] and up to 11 by MALDI-TOF.[14] Thiolysis of extracts followed by gel permeation chromatography identified PAs with DPs of up to 18[8,15]; in direct-infusion electrospray ionization spectra, [M 3H]3 signals of PAs up to 28 units were observed.[16] In general, MS does not clearly reveal high molecular weight (MW) compounds, mainly because of ion suppression caused by the high abundance of lower MW species.[11,17–19] The positive MALDI spectra of grape seed raw extracts show the signals of many putative PA oligomers, including those of several isobaric sodium and potassium adducts with the same nominal MW. Consequently, the MALDI-TOF profiles of samples show several signals that can be potentially assigned to different PAs. In this study, sodium and potassium exchange experiments on grape seed extracts were carried out to study the presence of
J. Mass Spectrom. 2014, 49, 826–830
isobaric signals corresponding to different PAs in the MALDI mass spectra. Extracts of six grape varieties were examined.
Materials and methods Samples and sample preparation Seed extracts of six grape varieties were studied: Aramon (Vitis vinifera × unknown variety), Bacò 1 (V. vinifera × V. riparia), Bertille Seyve 4825 (Bertille Seyve 2667 × Seibel 6905), Galibert 238-35 (Seyve Villard 12-401 × V. vinifera), Seibel 8357 (Seibel 6150 × Seibel 5455) and Seyve Villard 12-390 (Seibel 6468 × Seibel 6905). Grape samples were collected in 2010 at full technological ripeness (maximum sugar content) from the five plants of each variety present in the CRA – Viticulture Research Centre Grapevine Germplasm Collection (Susegana, Treviso, Italy) and immediately frozen. Seeds of 20 berries were manually separated from skins and pulps, dried on a paper sheet, weighted and ground with liquid nitrogen by using a mortar. The sample was defatted with hexane (ratio 1:10 w/v) by keeping the sample under stirring at room temperature overnight in the dark. The powder was separated from the solvent by a paper filter and extracted with a methanol/water solution (70 : 30 v/v) in ratio 1 : 10 (w/v).[20] A volume of 1 ml of the extract was reduced to 0.5 ml under vacuum, added with 4.5 ml water then passed through a 360-mg
* Correspondence to: Riccardo Flamini, Consiglio per la Ricerca e la Sperimentazione in Agricoltura – Centro di Ricerca per la Viticoltura (CRA-VIT), Viale XXVIII Aprile 26, 31015 Conegliano, Treviso, Italy. E-mail: riccardo.fl[email protected] †
This article is part of the Journal of Mass Spectrometry special issue entitled “3rd MS Food Day” edited by Gianluca Giorgi.
a CRA-VIT, Viale XXVIII aprile 26, 31015 Conegliano, Treviso, Italy b CNR-IENI, Corso Stati Uniti 4, 35127 Padova, Italy
Copyright © 2014 John Wiley & Sons, Ltd.
Isobaric grape proanthocyanidins by MALDI-TOF MS (70 : 30 v/v). The raw and purified extract solutions were diluted ten times with a methanol/water solution (70 : 30 v/v). In this study, 5 μl of these solutions were mixed with the same volume of matrix solution, and 1 μl of the resulting mixture was deposited directly on the stainless steel sample holder and allowed to dry before introduction into the mass spectrometer. External mass calibration (peptide calibration standard) was based on monoisotopic values of [M + H]+ of bradykinin, angiotensin II, angiotensin I, substance P, bombesin, ACTH clip (1–17), ACTH clip (18–39) and somatostatin 28 at m/z 757.39916, 1046.5420, 1296.6853, 1347.7361, 1619.8230, 2093.0868, 2465.1990 and 3147.4714, respectively. To perform cation exchanging experiments, water solutions of LiCl, NaCl and KCl at a 100-mM concentration were prepared. In this study, 2 μl of the cation salt solution was added to 5 μl of
Figure 1. Structures of grape seed flavanols and proanthocyanidins.
C18 Sep-Pak cartridge (Waters) previously activated by passage of 3 ml methanol and 5 ml water for removing salts and more polar compounds. After sample passage, the cartridge was washed with 3 ml of water, and the purified polyphenolic fraction was eluted with 3 ml of methanol.
Table 1. Peaks of putative isobaric pairs of PAs in the m/z 900–2000 range m/z
Compound
905
Dimer 2G Trimer Trimer 2G Tetramer Trimer 3G Tetramer 1G Tetramer 2G Pentamer Tetramer 3G Pentamer 1G Tetramer 4G Pentamer 2G Pentamer 2G Hexamer Pentamer 3G Hexamer 1G
1193 1345
MALDI-TOF MS
1481
Mass spectrometric measurements were performed using a MALDITOF-TOF UltrafleXtreme (Bruker Daltonics, Bremen, Germany) instrument, equipped with 1-kHz smartbeam II laser (λ = 355 nm) and operating in reflectron positive ion mode. The instrumental conditions employed to analyze molecular species in the m/z range 450–3500 were ion source 1: 25.00 kV; ion source 2: 22.30 kV; lens: 7.70 kV; pulsed ion extraction: 80 ns, reflector: 26.45 kV and reflector 2: 13.45 kV. The matrix solution was prepared by dissolving 10 mg of crystalline 2, 5-dihydroxybenzoic acid in 1 ml of methanol/water solution
1633 1785 1769 1921
Cation +
[M + Na] + [M + K] + [M + Na] + [M + K] + [M + Na] + [M + K] + [M + Na] + [M + K] + [M + Na] + [M + K] + [M + Na] + [M + K] + [M + Na] + [M + K] + [M + Na] + [M + K]
827
Figure 2. MALDI mass spectrum of Bacò 1 grape seeds raw extract.
J. Mass Spectrom. 2014, 49, 826–830
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jms
F. De Marchi et al. the solution containing the purified extract and stirred for about 1 min. After stirring, 5 μL of 2,5-dihydroxybenzoic acid solution was added, and 1 μl of the resulting mixture was deposited directly on the stainless steel sample holder and allowed to dry before introduction into the mass spectrometer.
Results and discussion The positive MALDI-TOF ‘fingerprints’ of grape seed extracts show decreasing peak intensities with increasing m/z values, corresponding to PAs [M + Na]+ and [M + K]+ adducts according to published
data (Fig. 2).[14,21] The signals of PA sodium and potassium adducts with the same nominal MW were identified among the peaks. In general, the more intense peaks observed at m/z
